Proteasome inhibitors and methods of using the same

ABSTRACT

The present invention provides boronic acid compounds, boronic esters, and compositions thereof that can modulate apoptosis such as by inhibition of proteasome activity. The compounds and compositions can be used in methods of inducing apoptosis and treating diseases such as cancer and other disorders associated directly of indirectly with proteasome activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/495,364, filed Aug. 14, 2003, the disclosure of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to boronic acid and boronic estercompounds useful as proteasome inhibitors and modulation of apoptosis.

BACKGROUND OF THE INVENTION

The proteasome, (also refered to as multicatalytic protease (MCP),multicatalytic proteinase, multicatalytic proteinase complex,multicatalytic endopeptidase complex, 20S, 26S, or ingensin) is a large,multiprotein complex present in both the cytoplasm and the nucleus ofall eukaryotic cells. It is a highly conserved cellular structure thatis responsible for the ATP-dependent proteolysis of most cellularproteins (Tanaka, Biochem Biophy. Res. Commun., 1998, 247, 537). The 26Sproteasome consists of a 20S core catalytic complex that is capped ateach end by a 19S regulatory subunit. The archaebacterial 20S proteasomecontains fourteen copies of two distinct types of subunits, α and β,which form a cylindrical structure consisting of four stacked rings. Thetop and bottom rings contain seven α-subunits each, while the innerrings contain seven β-subunits. The more complex eukaryotic 20Sproteasome is composed of about 15 distinct 20-30 kDa subunits and ischaracterized by three major activities with respect to peptidesubstrates. For example, the proteasome displays tryptic-,chymotryptic-, and peptidylglutamyl peptide-hydrolytic activities(Rivett, Biochem. J., 1993, 291, 1 and Orlowski, Biochemisty, 1990, 29,10289). Further, the proteasome has a unique active site mechanism whichis believed to utilize a threonine residue as the catalytic nucleophile(Seemuller, et al., Science, 1995, 268, 579).

The 26S proteasome is able to degrade proteins that have been marked bythe addition of ubiquitin molecules. Typically, ubiquitin is attached tothe ε-amino groups of lysines in a multistep process utilizing ATP andE1 (ubiquitin activating) and E2 (ubiquitin-conjugating) enzymes.Multi-ubiquitinated substrate proteins are recognized by the 26Sproteasome and are degraded. The multi-ubiquitin chains are generallyreleased from the complex and ubiquitin is recycled (Goldberg, et al.,Nature, 1992, 357, 375).

Numerous regulatory proteins are substrates for ubiquitin dependentproteolysis. Many of these proteins function as regulators ofphysiological as well as pathophysiological cellular processes.Alterations in proteasome activity have been implicated in a number ofpathologies including neurodegenerative diseases such as Parkinson'sdisease, Alzheimer's disease, as well as occlusion/ischaemia reperfusioninjuries, and aging of the central nervous system.

The ubiquitin-proteasome pathway also plays a role in neoplastic growth.The regulated degradation of proteins such as cyclins, CDK2 inhibitors,and tumor suppressors is believed to be important in cell cycleprogression and mitosis. A known substrate of the proteasome is thetumor suppressor p53 which is involved in several cellular processes(see, e.g., Ko, L. J. Genes Dev., 1996, 10, 1054). Tumor suppressor p53has been shown to induce apoptosis in several haematopoietic cell lines(Oren, M., Semin. Cancer Biol., 1994, 5, 221). Induction of p53 leads tocell growth arrest in the G1 phase of the cell cycle as well as celldeath by apoptosis. Tumor suppressor p53 degradation is known to becarried out via the ubiquitin-proteasome pathway, and disrupting p53degradation by inhibition of the proteasome is a possible mode ofinducing apoptosis.

The proteasome is also required for activation of the transcriptionfactor NF-κB by degradation of its inhibitory protein, IκB (Palombella,et al., Cell, 1994, 78, 773). NF-κB has a role in maintaining cellviability through the transcription of inhibitors of apoptosis. Blockadeof NF-κB activity has been demonstrated to make cells more susceptibleto apoptosis.

Several inhibitors of the proteolytic activity of the proteasome havebeen reported. See, for example, Kisselev, et al., Chemistry & Biology,2001, 8, 739. Lactacystin is a Streptomyces metabolite that specificallyinhibits the proteolytic activity of the proteasome complex (Fenteany,et al., Science, 1995, 268, 726). This molecule is capable of inhibitingthe proliferation of several cell types (Fenteany, et al., Proc. Natl.Acad. Sci. USA, 1994, 91, 3358). It has been shown that lactacystinbinds irreversibly, through its β-lactone moiety, to a threonine residuelocated at the amino terminus of the β-subunit of the proteasome.

Peptide aldehydes have been reported to inhibit the chymotrypsin-likeactivity associated with the proteasome (Vinitsky, et al., Biochemistry,1992, 31, 9421; Tsubuki, et al., Biochem. Biophys. Res. Commun., 1993,196, 1195; and Rock, et al., Cell, 1994, 78, 761). Dipeptidyl aldehydeinhibitors that have IC₅₀ values in the 10-100 nM range in vitro (Iqbal,M., et al., J. Med.Chem. 1995, 38, 2276) have also been reported. Aseries of similarly potent in vitro inhibitors from α.-ketocarbonyl andboronic ester derived dipeptides has also been reported (Iqbal, et al.,Bioorg. Med. Chem. Lett. 1996, 6, 287, U.S. Pat. Nos. 5,614,649;5,830,870; 5,990,083; 6,096,778; 6,310,057; U.S. Pat. App. Pub. No.2001/0012854, and WO 99/30707).

N-terminal peptidyl boronic ester and acid compounds have been reportedpreviously (U.S. Pat. Nos. 4,499,082 and 4,537,773; WO 91/13904;Kettner, et al., J. Biol. Chem., 1984, 259, 15106). These compounds arereported to be inhibitors of certain proteolytic enzymes. N-terminaltri-peptide boronic ester and acid compounds have been shown to inhibitthe growth of cancer cells (U.S. Pat. No. 5,106,948). A broad class ofN-terminal tri-peptide boronic ester and acid compounds and analogsthereof has been shown to inhibit renin (U.S. Pat. No. 5,169,841).

Various inhibitors of the peptidase activities of the proteasome havealso been reported. See, e.g., Dick, et al., Biochemistry, 1991, 30,2725; Goldberg, et al., Nature, 1992, 357, 375; Goldberg, Eur. J.Biochem., 1992, 203, 9; Orlowski, Biochemistry, 1990, 29, 10289; Rivett,et al., Archs. Biochem. Biophys., 1989, 218, 1; Rivett, et al., J. Biol.Chem., 1989, 264, 12215; Tanaka, et al., New Biol., 1992, 4, 1;Murakami, et al., Proc. Natl. Acad Sci. USA, 1986, 83, 7588; Li et al.,Biochemistry, 1991, 30, 9709; Goldberg, Eur. J. Biochem., 1992, 203, 9;and Aoyagi, et al., Proteases and Biological Control, Cold Spring HarborLaboratory Press (1975), pp. 429-454.

Stein et al., U.S. patent application Ser. No. 08/212,909, filed Mar.15, 1994, report peptide aldehydes useful for reducing in an animal boththe rate of loss of muscle mass and the rate of intracellular proteinbreakdown. The compounds are also said to reduce the rate of degradationof p53 protein in an animal. Palombella, et al., WO 95/25533, report theuse of peptide aldehydes to reduce the cellular content and activity ofNF-κB in an animal by contacting cells of the animal with a peptidealdehyde inhibitor of proteasome function or ubiquitin conjugation.Goldberg and Rock, WO 94/17816, report the use of proteasome inhibitorsto inhibit MHC-I antigen presentation. Stein, et al., U.S. Pat. No.5,693,617 report peptidyl aldehyde compounds as proteasome inhibitorsuseful for reducing the rate of degradation of protein in an animal.Inhibition of the 26S and 20S proteasome by indanone derivatives and amethod for inhibiting cell proliferation using indanone derivatives arereported by Lum et al., U.S. Pat. No. 5,834,487. Alpha-ketoamidecompounds useful for treating disorders mediated by 20S proteasome inmammals are reported in Wang et al., U.S. Pat. No. 6,075,150. France, etal., WO 00/64863, report the use of 2,4-diamino-3-hydroxycarboxylic acidderivatives as proteasome inhibitors. Carboxylic acid derivatives asproteasome inhibitors are reported by Yamaguchi et al., EP 1166781.Ditzel, et al., EP 0 995 757 report bivalent inhibitors of theproteasome. 2-Aminobenzylstatine derivatives that inhibit non-covalentlythe chymotrypsin-like activity of the 20S proteasome have been reportedby Garcia-Echeverria, et al., Bioorg. Med. Chem. Lett., 2001, 11, 1317.

Some further proteasome inhibitors can contain boron moieties. Forexample, Drexler et al., WO 00/64467, report a method of selectivelyinducing apoptosis in activated endothelial cells or leukemic cellshaving a high expression level of c-myc by using tetrapeptidic boronatecontaining proteasome inhibitors. Furet et al., WO 02/096933 report2-[[N-(2-amino-3-(heteroaryl oraryl)propionyl)aminoacyl]amino]-alkylboronic acids and esters for thetherapeutic treatment of proliferative diseases in warm-blooded animals.U.S. Pat. Nos. 6,083,903; 6,297,217; 5,780454; 6,066,730; 6,297,217;6,548,668; U.S. Patent Application Pub. No. 2002/0173488; and WO96/13266 report boronic ester and acid compounds and a method forreducing the rate of degradation of proteins. A method for inhibitingviral replication using certain boronic acids and esters is alsoreported in U.S. Pat. No. 6,465,433 and WO 01/02424. Pharmaceuticallyacceptable compositions of boronic acids and novel boronic acidanhydrides and boronate ester compounds are reported by Plamondon, etal., U.S. Patent Application Pub. No. 2002/0188100. A series of di- andtripeptidyl boronic acids are shown to be inhibitors of 20S and 26Sproteasome in Gardner, et al., Biochem. J., 2000, 346, 447.

Other boron-containing peptidyl and related compounds are reported inU.S. Pat. Nos. 5,250,720; 5,242,904; 5,187,157; 5,159,060; 5,106,948;4,963,655; 4,499,082; and WO 89/09225, WO/98/17679, WO 98/22496, WO00/66557, WO 02/059130, WO 03/15706, WO 96/12499, WO 95/20603, WO95/09838, WO 94/25051, WO 94/25049, WO 94/04653, WO 02/08187, EP 632026,and EP 354522.

A great interest exists, as evidenced by the above references, in drugswhich can modulate proteasome activity. For example, molecules capableof inhibiting proteasome activity can arrest or delay cancer progressionby interfering with the ordered degradation of cell cycle proteins ortumor suppressors. Accordingly, there is an ongoing need for new and/orimproved inhibitors of proteasome.

SUMMARY OF THE INVENTION

The present invention is directed to novel boronic acid and boronicester compounds useful as proteasome inhibitors and modulation ofapoptosis. The subject invention also comprises methods for inhibitionof multicatalytic protease (“MCP”) associated with certain disorders,including the treatment of muscle wasting disorders.

In one embodiment are provided compounds having Formula (I):

wherein constituent members are defined infra, as well as preferredconstituent members.

In another embodiment the present invention provides a pharmaceuticalcomposition comprising a compound of Formula (I) and a pharmaceuticallyacceptable carrier.

In another embodiment the present invention provides a method ofinhibiting activity of proteasome comprising contacting a compound ofFormula (I) with said proteasome.

In another embodiment the present invention provides a method oftreating cancer comprising administering to a mammal having orpredisposed to said cancer a therapeutically effective amount of acompound of Formula (I).

In another embodiment the present invention provides a method oftreating cancer comprising administering to a mammal having orpredisposed to said cancer a therapeutically effective amount of acompound of Formula (I), and wherein said cancer is selected from skin,prostate, colorectal, pancreas, kidney, ovary, mammary, liver, tongue,lung, and smooth muscle tissue.

In another embodiment the present invention provides a method oftreating cancer comprising administering to a mammal having orpredisposed to said cancer a therapeutically effective amount of acompound of Formula (I), and wherein said cancer is selected fromleukemia, lymphoma, non-Hodgkin lymphoma, myeloma, and multiple myeloma.

In another embodiment the present invention provides a method oftreating cancer comprising administering to a mammal having orpredisposed to said cancer a therapeutically effective amount of acompound of Formula (I) in combination with one or more antitumor oranticancer agent and/or radiotherapy.

In another embodiment the present invention provides a method ofinhibiting activity of transcription factor NF-κB comprising contactingIκB, the inhibitor of transcription factor NF-κB, with a compound ofFormula (I).

These and other features of the compounds will be set forth in expandedform as the disclosure continues.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention provides, inter alia, compounds that can inhibitproteasome activity and can be used for the treatment of diseases ordisorders related to proteasome activity. Compounds of the inventioninclude compounds of Formula (I):

-   -   or pharmaceutically acceptable salts, stereoisomeric or        tautomeric forms thereof, wherein:    -   R¹ is C₁-C₄ alkyl;    -   Y is or H, CN, NO₂, or a guanidino protecting group;    -   Z is —CN, —C(═O)OR⁵, phthalimido, —NHSO₂R⁶, or —NR³R⁴;    -   R³ is H or C₁-C₄ alkyl;    -   R⁴ is selected from the group aryl-SO₂—, aryl-C(═O)—,        aralkyl-C(═O)—, alkyl-C(═O)—, aryl-OC(═O)—, aralkyl-OC(═O)—,        alkyl-OC(═O)—, aryl-NHC(═O)—, and alkyl-NHC(═O)—; wherein aryl        or aralkyl is optionally substituted by one or more methyl or        methoxy groups;    -   R⁵ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, wherein R⁵        is optionally substituted by one or more halo, C₁-C₄ alkyl,        aryl, or heteroaryl;    -   R⁶ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, wherein R⁶ is        optionally substituted by one or more halo, C₁-C₄ alkyl, aryl,        or heteroaryl;    -   Q is selected from B(OR⁹)₂, pinanediol boronic ester, pinacol        boronic ester, 1,2-ethanediol boronic ester, 1,3-propanediol        boronic ester, 1,2-propanediol boronic ester, 2,3-butanediol        boronic ester, 1,1,2,2-tetramethylethanediol boronic ester,        1,2-diisopropylethanediol boronic ester, 5,6-decanediol boronic        ester, 1,2-dicyclohexylethanediol boronic ester,        bicyclohexyl-1,1′-diol boronic ester, and        1,2-diphenyl-1,2-ethanediol boronic ester;    -   R⁹ is H, C₁-C₄ alkyl, cycloalkyl, cycloalkylalkyl, aryl, or        aralkyl;    -   m is 2, 3, or 4; and    -   n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

In some embodiments R¹ is ethyl, propyl, or butyl.

In some embodiments R¹ is 2-propyl.

In some embodiments Q is pinanediol boronic ester.

In some embodiments Q is bicyclohexyl-1,1′-diol boronic ester.

In some embodiments Q is B(OH)₂.

In some embodiments Z is phthalimido, NHR⁴, or CN.

In some embodiments n is 8, 9, 10, or 11.

In some embodiments m is 3.

In some embodiments n is 8, 9, 10, or 11; and m is 3.

In some embodiments the present invention provides compounds Formula(I):

-   -   or pharmaceutically acceptable salys, stereoisomeric or        tautomeric forms thereof, wherein:    -   R¹ is 2-propyl;    -   Y is —NO₂;    -   Z is —CN, —C(═O)OR⁵, phthalimido, —NHSO₂R⁶, or —NHR⁴;    -   R⁴ is selected from the group aryl-SO₂—, alkyl-SO₂—,        aryl-C(═O)—, aralkyl-C(═O)—, alkyl-C(═O)—, aryl-OC(═O)—,        aralkyl-OC(═O)—, alkyl-OC(═O)—, aryl-NHC(═O)—, and        alkyl-NHC(═O)—; wherein said aryl or aralkyl is optionally        substituted by one or more methyl or methoxy groups;    -   R⁵ is H or C₁-C₆ alkyl, wherein said alkyl is optionally        substituted with one or more halo, aryl, or heteroaryl;    -   R⁶ is C₁-C₆ alkyl, wherein said alkyl is optionally substituted        with one or more halo, aryl, or heteroaryl;    -   Q is selected from B(OR⁹)₂, pinanediol boronic ester, pinacol        boronic ester, 1,2-ethanediol boronic ester, 1,3-propanediol        boronic ester, 1,2-propanediol boronic ester, 2,3-butanediol        boronic ester, 1,1,2,2-tetramethylethanediol boronic ester,        1,2-diisopropylethanediol boronic ester, 5,6-decanediol boronic        ester, 1,2-dicyclohexylethanediol boronic ester,        bicyclohexyl-1,1′-diol boronic ester, and        1,2-diphenyl-1,2-ethanediol boronic ester;    -   R⁹ is H or C₁-C₄ alkyl;    -   m is 3; and    -   n is 4, 5, 6, 7, 8, 9, 10 or 11.

In some embodiments the present invention provides compounds of Formula(I-s):

-   -   or a pharmaceutically acceptable salts, stereoisomeric or        tautomeric forms thereof, wherein:    -   R¹ is C₁-C₄ alkyl;    -   Y is CN or NO₂;    -   Z is —CN, —C(═O)OR⁵, phthalimido, —NHSO₂R⁶, or —NR³R⁴;    -   R³ is H or C₁-C₄ alkyl;    -   R⁴ is selected from the group aryl-SO₂—, alkyl-SO₂—,        aryl-C(═O)—, aralkyl-C(═O)—, alkyl-C(═O)—, aryl-OC(═O)—,        aralkyl-OC(═O)—, alkyl-OC(═O)—, aryl-NHC(═O)—, and        alkyl-NHC(═O)—; wherein said aryl or aralkyl is optionally        substituted by one or more methyl or methoxy groups;    -   R⁵ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, wherein R⁵        is optionally substituted by one or more halo, C₁-C₄ alkyl,        aryl, or heteroaryl;    -   R⁶ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, wherein R₆ is        optionally substituted by one or more halo, C₁-C₄ alkyl, aryl,        or heteroaryl;    -   Q is selected from B(OR⁹)₂, pinanediol boronic ester,        1,2-dicyclohexylethanediol boronic ester, and        bicyclohexyl-1,1′-diol boronic ester;    -   R⁹ is H or C₁-C₄ alkyl;    -   m is 2, 3, or 4; and    -   n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

In some embodiments the present invention provides compounds of Formula(I-s):

or pharmaceutically acceptable salt forms thereof.

In yet further embodiments the present invention provides compounds ofFormula (I-s) selected from: Example 10, Example 11, Example 12, Example13, Example 14, Example 15, Example 16, Example 17, Example 18, Example19, Example 20, Example 21, Example 22, Example 23, and Example 24; or apharmaceutically acceptable salt or free base form thereof.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

As used herein, the phrase “boronic acid” refers to a compoundcontaining a B(OH)₂ moiety. In some embodiments, boronic acid compoundscan form oligomeric anhydrides by dehydration of the boronic moiety. Forexample, Snyder, et al., J. Am. Chem. Soc., 1958, 80, 3611 reportoligomeric arylboronic acids. Thus, unless otherwise indicated, “boronicacid”, or a chemical Formula containing a —B(OH)₂ moiety, is intended toencompass free boronic acids, oligomeric anhydrides, including but notlimited to, dimers, trimers, tetramers, and mixtures thereof.

As used herein, “boronic acid anhydride” or “boronic anhydride” refersto a compound formed by the combination of two or more molecules of aboronic acid compound of Formula (I), with loss of one or more watermolecules from the boronic acid moieties. When contacted with water, theboronic acid anhydride compound can be hydrated to release free boronicacid compound. In some embodiments, the boronic acid anhydride structurecan contain two, three, four, or more boronic acid units and can have acyclic or linear configuration. In some embodiments, the boronic acidanhydride compound exists substantially in a single oligomeric form;however, boronic acid anhydrides also encompass mixtures of differentoligomeric boronic acid anhydride as well as free boronic acids.

Non-limiting examples of boronic acid anhydrides of the inventioninclude compounds of Formula (II) and (III) where G is a moiety ofFormula (IV) and t is 0 to 10 or 1, 2, 3, or 4.

In some embodiments, at least about 80% of boronic acid present in aboronic acid anhydride compound exists in a single oligomeric anhydrideform. In further embodiments, at least about 85, about 90, about 95, orabout 99% of the boronic acid present in the boronic acid anhydrideexists in a single oligomeric anhydride form. In some embodiments, theboronic acid anhydride compound consists essentially of a singleoligomeric boronic acid anhydride. In yet further embodiments, theboronic acid anhydride compound consists of a single oligomeric boronicacid anhydride. In further embodiments, the boronic acid anhydridecompound contains a boroxine for Formula (III), wherein t is 1.

Boronic acid anhydride compounds can be prepared from the correspondingboronic acid compound by exposure to dehydrating conditions, including,for example, crystallization, lyophilization, exposure to heat, and/orexposure to a drying agent. Some suitable crystallization solventsinclude ethyl acetate, dichloromethane, hexanes, ether, benzene,acetonitrile, ethanol, and mixtures thereof.

As used herein, the phrase “boronic ester” or boronic acid ester” refersto an ester derivative of a boronic acid compound.

As used herein, “cyclic boronic ester” is intended to mean a stablecyclic boronic moiety of general formula —B(OR)(OR) wherein the two Rsubstituents taken together contain from 2 to 20 carbon atoms, andoptionally, a heteroatom which can be N, S, or O. Cyclic boronic estersare well known in the art. Examples of cyclic boronic ester include, butare not limited to, pinanediol boronic ester, pinacol boronic ester,1,2-ethanediol boronic ester, 1,3-propanediol boronic ester,1,2-propanediol boronic ester, 2,3-butanediol boronic ester,1,1,2,2-tetramethylethanediol boronic ester, 1,2-diisopropylethanediolboronic ester, 5,6-decanediol boronic ester, 1,2-dicyclohexylethanediolboronic ester, bicyclohexyl-1,1′-diol, diethanolamine boronic ester, and1,2-diphenyl-1,2-ethanediol boronic ester.

As used herein, the term “alkyl” or “alkylene” is meant to refer to asaturated hydrocarbon group which is straight-chained or branched.Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g.,n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl,t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl) and the like. Analkyl group can contain from 1 to about 20, from 2 to about 20, from 1to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, orfrom 1 to about 3 carbon atoms.

As used herein, “alkenyl” refers to an alkyl group having one or moredouble carbon-carbon bonds. Example alkenyl groups include ethenyl,propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl,hexadienyl, and the like.

As used herein, “alkynyl” refers to an alkyl group having one or moretriple carbon-carbon bonds. Example alkynyl groups include ethynyl,propynyl, butynyl, pentynyl, and the like.

As used herein, “haloalkyl” refers to an alkyl group having one or morehalogen substituents. Example haloalkyl groups include CF₃, C₂F₅, CHF₂,CCl₃, CHCl₂, C₂Cl₅, and the like. An alkyl group in which all of thehydrogen atoms are replaced with halogen atoms can be referred to as“perhaloalkyl.” Examples perhaloalkyl groups include CF₃ and C₂F₅.

As used herein, “carbocyclyl” groups are saturated (i.e., containing nodouble or triple bonds) or unsaturated (i.e., containing one or moredouble or triple bonds) cyclic hydrocarbon moieties. Carbocyclyl groupscan be mono- or polycyclic. Example carbocyclyl groups includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, norbornyl, norpinyl,norcarnyl, adamantyl, phenyl, and the like. Carbocyclyl groups can bearomatic (e.g., “aryl”) or non-aromatic (e.g., “cycloalkyl”). In someembodiments, carbocyclyl groups can have from 3 to about 20, 3 to about10, or 3 to about 7 carbon atoms.

As used herein, “aryl” refers to aromatic carbocyclyl groups includingmonocyclic or polycyclic aromatic hydrocarbons such as, for example,phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like. In someembodiments, aryl groups have from 6 to about 18 carbon atoms.

As used herein, “cycloalkyl” refers to non-aromatic carbocyclyl groupsincluding cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groupcan include bi- or poly-cyclic ring systems. Example cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl,norbornyl, norpinyl, norcarnyl, adamantyl, and the like. Also includedin the definition of cycloalkyl are moieties that have one or morearomatic rings fused (i.e., having a bond in common with) to thecycloalkyl ring, for example, benzo derivatives of cyclopentane(indanyl), cyclohexane (tetrahydronaphthyl), and the like.

As used herein, “heterocarbocyclyl” groups can be saturated orunsaturated carbocyclyl groups wherein one or more of the ring-formingcarbon atoms of the carbocyclyl group is replaced by a heteroatom suchas O, S, or N. Heterocarbocyclyl groups can be aromatic (e.g.,“heteroaryl”) or non-aromatic (e.g., “heterocycloalkyl”).Heterocarbocyclyl groups can correspond to hydrogenated and partiallyhydrogenated heteroaryl groups. Heterocarbocyclyl groups can contain, inaddition to at least one heteroatom, from about 1 to about 20, about 2to about 10, or about 2 to about 7 carbon atoms and can be attachedthrough a carbon atom or heteroatom. Further, heterocarbocyclyl groupscan be substituted or unsubstituted. Examples of heterocarbocyclylgroups include morpholino, thiomorpholino, piperazinyl,tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl,1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl,isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl,thiazolidinyl, imidazolidinyl, and the like.

As used herein, “heteroaryl” groups are aromatic heterocarbocyclylgroups and include monocyclic and polycyclic aromatic hydrocarbons thathave at least one heteroatom ring member such as sulfur, oxygen, ornitrogen. Heteroaryl groups include, without limitation, pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl,isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl,oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl,pyrazolyl, triazolyl, tetrazolyl indazolyl, 1,2,4-thiadiazolyl,isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, and thelike. In some embodiments, heteroaryl groups can have from 1 to about 20carbon atoms, and in further embodiments from about 3 to about 20 carbonatoms. In some embodiments, heteroaryl groups have 1 to about 4, 1 toabout 3, or 1 to 2 heteroatoms.

As used herein, “heterocycloalkyl” refers to a non-aromaticheterocarbocyclyl group including cyclized alkyl, alkenyl, and alkynylgroups where one or more of the ring-forming carbon atoms is replaced bya heteroatom such as an O, N, or S atom. Also included in the definitionof heterocycloalkyl are moieties that have one or more aromatic ringsfused (i.e., having a bond in common with) to the nonaromaticheterocyclic ring, for example phthalimidyl, naphthalimidyl pyromelliticdiimidyl, phthalanyl, and benzo derivatives of saturated heterocyclessuch as indolene and isoindolene groups.

As used herein, “halo” or “halogen” includes fluoro, chloro, bromo, andiodo.

As used herein, “alkoxy” refers to an —O-alkyl group. Example alkoxygroups include methoxy, ethoxy, propoxy (e.g., n-propoxy andisopropoxy), t-butoxy, and the like.

As used herein, “thioalkoxy” refers to an alkoxy group in which the Oatom is replaced by an S atom.

As used herein, “aryloxy” refers to an —O-aryl group. An example aryloxygroup is phenoxy.

As used herein, “aralkyl” refers to an alkyl moiety substituted by anaryl group. Example aralkyl groups include benzyl and naphthylmethylgroups. In some embodiments, arylalkyl groups have from 7 to 11 carbonatoms.

As used herein, “amino” refers to an NH₂ group. “Alkylamino” refers toan amino group substituted by an alkyl group and “dialkylamino” refersto an amino group substituted by two alkyl groups. On the contrary,“aminoalkyl” refers to an alkyl group substituted by an amino group.

As used herein, “cycloalkylalkyl” refers to an alkyl group substitutedby a cycloalkyl group.

As used herein, the phrase “protecting group” refers to a chemicalfunctional group that can be selectively appended to and removed fromfunctionalities, such as hydroxyl groups, amino groups, and carboxylgroups. Protecting groups are usually introduced into a chemicalcompound to render such functionality inert to chemical reactionconditions to which the compound is exposed. Any of a variety ofprotecting groups can be employed with the present invention. Aprotecting group of an amino moiety can be referred to as an “aminoprotecting group” and a protecting group of a guanidino moiety can bereferred to as a “guanidino protecting group.” Amino and guanidinoprotecting groups can have the formulas aryl-SO₂—, alkyl-SO₂—,aryl-C(═O)—, aralkyl-C(═O)—, alkyl-C(═O)—, aryl-OC(═O)—,aralkyl-OC(═O)—, alkyl-OC(═O)—, aryl-NHC(═O)—, alkyl-NHC(═O)—, and thelike, wherein said alkyl, aryl and aralkyl groups may be substituted orunsubstituted. Example amino and guanidino protecting groups can alsoinclude t-butyloxycarbonyl (BOC), fluorenylmethoxycarbonyl (Fmoc),benzyloxycarbonyl (Cbz), and a phthalimido group. Other protectinggroups include the following moieties:

Further representative protecting groups can be found in T. W. Green andP. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley &Sons, Inc., New York (1999), which is incorporated herein by referencein its entirety.

As used herein, “substituted” indicates that at least one hydrogen atomof a chemical group is replaced by a non-hydrogen moiety. Examplesubstituents include F, Cl, Br, I, C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆,alkynyl, haloalkyl, NRERF, N₃, NO₂, CN, CNO, CNS, C(═O)OR^(E), R^(E)CO,R^(E)C(═O)O, R^(E)CONR^(E), R^(E)R^(F)NCO, ureido, OR^(E), SR^(E),SO₂-alkyl, SO₂-aryl, and SO₂—NR^(E)R^(F), wherein R^(E) and R^(F) areeach, independently, H or C₁-C₆ alkyl Alternatively, R^(E) and R^(F) maybe combined, with the nitrogen to which they are attached, to form a 5to 7 membered heterocyclic ring, for example pyrrolidinyl, piperidinyl,morpholinyl, piperazinyl, and N-methylpiperazinyl. When a chemical groupherein is “substituted” it may have up to the full valance ofsubstitution, provided the resulting compound is a stable compound orstable structure; for example, a methyl group may be substituted by 1,2, or 3 substituents, a methylene group may be substituted by 1 or 2substituents, a phenyl group may be substituted by 1, 2, 3, 4, or 5substituents, and the like.

As used herein “stable compound” or “stable structure” refers to acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and preferably capable offormulation into an efficacious therapeutic agent. The present inventionis directed only to stable compounds.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent invention that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis.

Compounds of the invention can also include tautomeric forms, such asketo-enol tautomers. Tautomeric forms can be in equilibrium orsterically locked into one form by appropriate substitution.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like. The pharmaceuticallyacceptable salts of the present invention can be synthesized from theparent compound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosureof which is hereby incorporated by reference.

Synthesis

Compounds of the invention, including salts and solvates thereof, can beprepared using known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes.

The reactions for preparing compounds of the invention can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Green and P. G. M.Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons,Inc., New York (1999), which is incorporated herein by reference in itsentirety.

Compounds of the invention can be prepared by the sequential coupling ofthree fragment components (F1, F2, and F3).

F1 Fragment

Synthesis of compounds of the invention can involve a boron-containingfragment (F1) having a structure indicated by Formula (A).

The boronic ester moiety of F1 can include, for example, a diol estersuch as is indicated by the loop connecting oxygen atoms in Formula (A).

Stereochemistry at the carbon atom alpha to the boron atom in Formula(A) can be controlled using an asymmetric boronic ester group in thepreparation of F1. For example, pinanediol esters of boronic acid canfacilitate the preparation of stereochemically pure, or substantiallystereochemically pure, F1 fragment. As an example, the F1 fragment canbe prepared by reacting a compound of Formula (B) (showing a pinanediolboronic ester obtained from (+)-pinanediol) with a strong base (e.g.,lithium diisopropylamide or lithium dicyclohexylamide) in the presenceof an excess of dichloromethane or dibromomethane, followed by additionof a Lewis acid, (e.g., ZnCl₂, ZnBr₂, or FeCl₃) to yield a compound ofFormula (C) (where L is halo) having a newly introduced stereocenter atthe carbon alpha to the boron.

The compound of Formula (B) can also be prepared according to a two stepprocedure involving reaction of a trialkoxyborane, preferablytriisopropoxyborane, with (1S, 2S, 3R, 5S)-(+) pinanediol, to give amono-alkoxy [(1S, 2S, 3R, 5S)-(+) pinanediol] borane intermediatewherein two of the alkoxy groups of the trialkoxy borane have beenreplaced by (1S, 2S, 3R, 5S)-(+) pinanediol. This mixed pinanediolalkoxy borane, upon reaction with the appropriate organometallicderivative, e.g. the Grignard reagent R¹CH₂MgBr or the alkyl lithiumR¹CH₂Li, gives compound (B) in good yields and purities. The processstarting from triisopropoxyborane to give the intermediate mixedpinanediol isopropoxy borane (F) and the compounds of formula (B) isdepicted in the following scheme below.

The compound of Formula (C) can, in turn, be reacted with an alkaliamide (e.g., lithium bis(trimethylsilyl)amide, sodiumbis(trimethylsilyl)amide, and potassium bis(trimethylsilyl)amide) orother nucleophile that effectively inverts the newly formed stereocenter(such as by an SN2 type mechanism) and introduces an amine group (NR₂)in place of the halo group (e.g., chloro), forming a compound of Formula(D) (where R can be, e.g., alkyl, Si(alkyl)₃, aryl, or aralkyl).

The compound of Formula (D) can be further reacted with an agent capableof converting the NR₂ group to NH₂, or salt thereof, to form an F1fragment substantially capable of coupling with a further fragmentthrough the amine. A suitable agent for converting the NR₂ group to NH₂can be a protic acid such as HCl such as when R is a silyl group (e.g.,trimethylsilyl).

F2 Fragment

The mid-section of compounds of the present invention can be representedby fragment F2 which couples to fragment F1 by peptide bond formationfor form an F2-F1 intermediate. Methods for coupling compounds throughpeptide bonds, or amide bonds, are well known in the art and described,for example, in The Peptides: Analysis, Synthesis, Biology, Vol. I.,eds. Gross, et al., Academic Press, 1979. An example F2 fragment isprovided in Formula (E) (Pg is an amino protecting group, Y and m aredefined herein). Additionally, protection of the amino group of aminoacids using Boc or other amino protecting groups is well known in theart.

Compounds of Formula (E) are amino acids or amino acid derivativesavailable commercially or prepared by routine methods known to oneskilled in the art.

F3 Fragments

A further fragment (F3) can be coupled to the F2 fragment of the F2-F1intermediate by peptide bond formation. Methods for coupling compoundsthrough peptide bonds, or amide bonds, are well known in the art anddescribed, for example, in The Peptides: Analysis, Synthesis, Biology,Vol. I., eds. Gross, et al., Academic Press, 1979. An example F2fragment is provided in Formula (F) (R² is H, and Z and n are definedherein)

Other coupling means are known in the art and are also suitable. F3fragments can be obtained from commercial sources or made by methodsknown to one skilled in the art.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C)infrared spectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Compounds of the invention can be prepared according to methods forpreparing aminoboronic acids, esters thereof, and related compoundsdescribed in the art, such as in U.S. Pat. Nos. 4,537,773 and 5,614,649,each of which is incorporated herein by reference in its entirety.

Compounds of the invention containing boronic esters, such as pinanediolesters, can be hydrolyzed by any suitable means to prepare correspondingboronic acid (—B(OH)₂) derivatives. Hydrolysis conditions can includecontacting a boronic ester with excess acid, such as a protic acid likeHCl.

Conversely, boronic acids can be esterified by contacting the acidcompound (—B(OH)₂) with an alcohol such as a diol for sufficient time toproduce the corresponding ester. The esterification reaction can be acidor base catalyzed.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES Example 1

Synthesis of(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutylaminehydrochloride salt

Step 1:2-(2-Methylpropyl)-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborole

A mixture of (+)-pinanediol (23.9 g, 0.140 mol) and2-methylpropylboronic acid (15 g, 0.147 mol) in diethyl ether (300 ml)was stirred at room temperature for 24 h. The mixture was dried overanhydrous sodium sulfate and purified by column chromatography (Silicagel 230-400 mesh), eluting with hexane:ethyl acetate 90:10 mixture. Theproduct was obtained as a clear oil (32.6 g, 94% yield).

¹H NMR (DMSO-d₆): 4.28 (1H, dd, J=8.8 Hz, 2.0); 2.30 (1H, m); 2.18 (1H,m); 1.96 (1H, t, J=5.3); 1.86 (1H, m); 1.78 (1H, set, J=6.8); 1.68 (1H,m); 1.30 (3H, s); 1.25 (3H, s); 1.01 (1H, d); 0.9 (6H, d, J=6.6 ); 0.81(3H,s); 0.69 (2H, m).Step 2:2-[(1S)-1-Chloro-3-methylbutyl]-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborole

A solution of lithium diisopropylamide was prepared by addition of 10.0M butyl lithium solution in hexane (25.4 ml, 0.254 mol) to a solution ofdiisopropylamine (35.7 ml, 0.254 mol) in dry tetrahydrofuran (60 ml), at−50 ° C., and allowing the temperature to rise to −30 ° C. This solutionwas transferred via canula into a solution of2-(2-methylpropyl)-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaboroleof Step 1 (50 g, 0.212 mol) and CH₂Cl₂ (50 ml, 0.848 mol) in drytetrahydrofuran (700 ml), while keeping the temperature below −70° C. A1.0 M solution of dry zinc chloride in diethyl ether (339 ml, 0.339 mol)was then added over a 30 minute period while keeping the internaltemperature below −70° C. The reaction mixture was stirred at −78° C.for 3 hours, then allowed to warm to room temperature. After removal ofthe solvents by rotary evaporation the residue was partitioned betweenpetroleum ether (1000 ml) and a 10% aqueous solution of ammoniumchloride (800 ml). The aqueous layer was further extracted withpetroleum ether (300 ml). The combined organic phases were dried overanhydrous sodium sulfate and concentrated. The product was obtained as abrown oil (59.0 g, 98% yield) containing about 9% mol/mol of startingmaterial (¹H-NMR), and was used in the subsequent step without furtherpurification.

¹H NMR (DMSO-d₆): 4.43 (1H, dd, J=8.8, 1.8); 3.59 (1H, m); 2.33 (1H, m);2.21 (1H, m); 2.01 (1H, m); 1.88 (1H, m); 1.84-1.55 (5H, m); 1.34 (3H,s); 1.26 (3H, s); 1.09 (1H, , J=10.1); 0.9 (3H, d, J=6.8); 0.87 (3H, d,J=6.4); 0.82 (3H, s).Step 3:N,N-Bis(trimethylsilyl)-(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutylamine

A 1.0 M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran(189 ml, 0.189 mol) was added, over 30 minutes, to a solution of crude2-[(1S)-1-chloro-3-methylbutyl]-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaboroleof Step 2 (59.0 g, 91% purity, 0.189 mol) in tetrahydrofuran (580 ml)while cooling at −78 ° C. The reaction mixture was allowed to slowlywarm to room temperature overnight. The solvent was removed by rotaryevaporation and the residue taken up with dry hexane (800 ml). Theresulting suspension was stirred at room temperature for 2 hours, thenthe solid was removed by filtration on a celite cake, which was washedwith dry hexane (3×100 ml). The filtrate was concentrated giving asatisfactorily pure product as a brown oil (79 g) in practicallyquantitative yield. The product was used for the subsequent step withoutfurther purification.

¹H NMR (DMSO-d₆): 4.33 (1H, dd, J=1.5 Hz, 8.6); 2.58 (1H, m); 2.29 (1H,m); 2.18 (1H, m); 1.95 (1H, t, J=5.9); 1.85 (1H, m); 1.9-1.55 (3H, m);1.31 (3H, s); 1.24 (3H, s); 1.17 (1H, m); 1.01 (1H, d, J=10.6); 0.85(3H, d, J=6.6), 0.83 (3H, d, J=6.6); 0.80 (18H, s).Step 4:(1R)-1-[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutylaminehydrochloride salt

To a solution of crudeN,N-Bis(trimethylsilyl)-(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutylamineof Step 3 (79 g, 0.193 mol) in a mixture of dioxane (100 ml) and diethylether (200 ml), a 4 N solution of hydrogen chloride in dioxane (193 ml,0.772 mol) was added, while cooling at 0° C. The mixture was thenstirred at room temperature for 4 hours and concentrated. The residuewas taken up with anhydrous hexane (500 ml) and a 2 M solution ofhydrogen chloride in diethyl ether (48 ml, 0.096 mol) was added. Themixture was stirred at 0° C. for 1 hour, then concentrated. The residuewas taken up with anhydrous hexane and the resulting suspension wasstirred at room temperature overnight. The solid was collected byfiltration and dried under vacuum affording 38.1 g of product (66%yield). A second crop (4.13 g, 7% yield) was obtained from the motherliquors.

¹H NMR (DMSO-d₆): 7.85 (3H, br); 4.45 (1H, dd, J=9.2 Hz); 2.78 (1H, m);2.34 (1H, m); 2.21 (1H, m); 2.01 (1H, t, J=5.3); 1.89 (1H, m); 1.82-1.65(2H, m); 1.49 (1H, m); 1.38 (3H, s); 1.27 (3H, s); 1.12 (1H, d, J=1.12);0.87 (6H, d, J=6.6); 0.83 (3H, s).

Example 2

Carbamic acid 1,1-dimethylethyl ester,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-.

Method A: HOAt/HATU

To a solution of BocNH(NO₂)ArgOH (15.7 g, 49.3 mmol) in anhydrous DMF(100 ml), HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; 18.7 g, 49.3 mmol) and HOAt(1-hydroxy-7-azabenzotriazole; 6.71 g, 49.3 mmol) were added. Themixture was cooled to 0° C. and N-methylmorpholine was added (13.6 ml,0.123 mol). After 10 minutes(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutylaminehydrochloride salt of Example 1 (12.4 g, 41.1 mmol) was added. Thecooling bath was removed and the mixture was stirred at r.t. for 4.5hours. The mixture was diluted with ethyl acetate (800 ml), washed witha 2% solution of citric acid (2×150 ml), 2% solution of NaHCO₃ (2×150ml) and 2% solution of NaCl (2×150 ml). The aqueous phases were furtherextracted with ethyl acetate (150 ml). The combined organic phases weredried over sodium sulfate and concentrated. The resulting oily residuewas redissolved in ethyl acetate (500 ml) and the solution was washedwith cold water (200 ml). The aqueous phases were further extracted withethyl acetate (500 ml). The combined organic phases were dried oversodium sulfate and concentrated. The residue was dissolved in diethylether (100 ml) an the solution was slowly added to hexane (600 ml) whilestirring. The white solid was collected by filtration (43.4 g) andpurified by column chromatography eluting initially with 50:50hexane:ethyl acetate mixture and then with ethyl acetate. The fractionscontaining the product were concentrated, the residue was dissolved indiethyl ether (100 ml) and the resulting solution was slowly added tohexane (600 ml) while stirring. The white solid was collected byfiltration (15.2 g, 66% yield).

Method B: IBCF

To a suspension of BocNH(NO₂)ArgOH (5.82 g, 18.2 mmol) in anhydrousdichloromethane (100 ml) N-methylmorpholine (2.0 ml, 18.2 mmol) wasadded. The mixture was cooled to −15° C. then isobutyl chloroformate wasadded (2.37 ml, 18.2 mmol). The mixture was stirred at −15° C. for 10minutes then(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutylaminehydrochloride salt obtained as in Example 1 was added (5.0 g, 16.6mmol), immediately followed by further N-methylmorpholine (2.0 ml, 18.2mmol). The reaction mixture was stirred for 1.5 hours at −15° C., thenallowed to warm to room temperature and partitioned between ethylacetate (150 ml), water (150 ml) and 0.1N hydrochloric acid (10 ml). Theorganic phase was washed with a saturated solution of NaHCO₃, dried overanhydrous sodium sulphate and concentrated. The oily residue (9.25 g)was purified by crystallization from ethyl acetate affording three cropsof satisfactorily pure product (5.03 g, 54% yield).

¹H NMR (DMSO-d₆): 8.80 (1H, br); 8.50 (1H, br), 7.87 (2H, br);7.01 (1H,d, J=7.9), 4.07 (1H, dd, J=7.9); 4.0 (1H, m); 3.12 (2H, m); 2.55 (1H,m); 2.2 (1H,m); 2.01 (1H, m); 1.83 (1H, t, J=5.1); 1.78 (1H, m);1.74-1.44 (7H, m); 1.38 (9H, s); 1.33 (1H, d, J=10.3); 1.24 (5H, s);1.22 (3H, s); 0.84 (6H, d, J=6.6); 0.81 (3H, s)

Example 3

(2S)-2-Amino-5-[[imino(nitroamino)methyl]amino]pentanamide,N-[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl];hydrochloride salt

Method A

A 4 N solution of hydrogen chloride in dioxane (15 ml) was added to asolution of carbamic acid 1,1-dimethylethyl ester,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-of Example 2, (4.04 g, 7.06 mmol) in a mixture of dioxane (40 ml) anddiethyl ether (7 ml), while cooling at 0° C. The reaction mixture wasallowed to warm to room temperature and stirred for further 4 hours. Thesolvent was removed by rotary evaporation, the residue was treated withdiethyl ether (50 ml) and the mixture was stirred at r.t. for threedays. The resulting solid was collected by filtration affording 3.18 gof pure product (90% yield)

Method B

Carbamic acid 1,1-dimethylethyl ester,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-of Example 2, (3 g, 5.3 mmol) was dissolved in Et₂O (40 mL) and asolution of about 10% HCl in Et₂O (20 mL) was added dropwise at 0° C.under nitrogen. The reaction mixture was allowed to warm to roomtemperature and to stir for further 5 hours.

The solvent was decanted and the residue, washed twice with Et₂O (20mL), was dried in vacuo to give the title compound as a white powder(2.43 g, yield 91%).

¹H NMR (DMSO-d₆): 8.56 (2H, br); 8.22 (3H, br);7.97 (2H, br); 4.28 (1H,dd, J=8.6 Hz, 2.01); 3.77 (1H, m); 3.04 (1H, m); 2.28 (1H, m); 2.11 (2H,m), 1.92 (1H, t, J=5.5); 1.83 (1H, m); 1.79-1.59 (4H, m); 1.59-1.37 (3H,m); 1.31 (4H, s); 1.24 (3H, s); 1.19 (1H, d, J=10.4); 0.88 (3H, d,J=6.0); 0.86 (3H, d, J=6.0); 0.81 (3H, s).

Example 4

Boronic acid,[(1R)-1-[[(2S)-2-amino-5-[[imino(nitroamino)methyl]amino]-1-oxopentyl]amino]-3-methylbutyl],hydrochloride salt

Carbamic acid 1,1-dimethylethyl ester,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-of Example 2, (3.1 g, 5.48 mmol) was carefully dissolved, under nitrogenat 0° C., in 20 mL of HCl 37%; the resultant mixture was allowed to warmto room temperature and to stir overnight. The reaction mixture waswashed with Et₂O until complete removal of pinanediol; the aqueoussolution was concentrated to dryness and dried in vacuo to afford 1.82grams (4.93 mmol, yield 90%) of the title compound, used without furtherpurification. ¹H-NMR: (DMSO+D₂O+TFA): 3.78(m, 1H); 3.19(m, 2H); 3.09(m,11H); 1.71(m, 2H); 1.70-1.48(m, 3H); 1.49-1.23(m, 2H); 0.89(d, J=5.8 Hz,3H); 0.88(d, J=5.8 Hz, 3H).

Example 5

10-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-decanoic acid.

Step 1: 2-Undec-10-enyl-1,3-dioxo-1,3-dihydroisoindole

To a mixture of 10-undecen-1-ol (4.23 g, 24.8 mmol), phthalimide (3.65g, 24.8 mmol) and triphenylphosphine (6.51 g, 24.8 mmol) in anhydroustetrahydrofuran (30 ml), a solution of DEAD (3.9 ml, 24.8 mmol) inanhydrous tetrahydrofuran (10 ml) was slowly added while keeping thetemperature below 8-10° C. After 2 hours further DEAD (1.0 ml, 6.37mmol) and triphenylphosphine (1.3 g, 4.96 mmol) were added and themixture was stirred at room temperature overnight. The reaction mixturewas concentrated and the residue was triturated with diethyl ether (50ml). The solid was removed by filtration and washed with diethyl ether(2×50 ml). The combined filtrates were concentrated and the residue wastriturated with hexane (50 ml) at 40° C. The resulting solid was removedby filtration and washed with hexane (2×50 ml). The combined filtrateswere concentrated and the residue was purified by column chromatographyeluting with 10:2 hexane:ethyl acetate mixture. The product was obtainedas a low-melting white solid (4.9 g, 66% yield). M.p. 25-30° C. ¹H NMR(DMSO-d₆) 7.83 (4H, m); 5.76 (1H, m); 4.96 (1H, dq, J=17.2, 1.6 Hz);4.90 (1H, ddt, J=10.2, 2.2, 1.1); 3.54 (2H, t, J=7.1), 1.97 (2H, q,J=6.7); 1.56 (2H, m); 1.35-1.15 (14H, m).

Step 2: 10-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-decanoic acid.

A solution of 2-undec-10-enyl-1,3-dioxo-1,3-dihydroisoindole (2 g, 6.68mmol) of Step 1 and Aliquatg 336 (0.2 g) in a mixture of hexane (20 ml)and acetic acid (6 ml) was added dropwise to a solution of potassiumpermanganate (2.76 g, 20 mmol) in water (28 ml) while cooling at 0° C.The reaction mixture was stirred at room temperature for 7 hours, thenan aqueous solution of sodium bisulfite was added until disappearance ofthe purple colour. The mixture was then extracted with ethyl acetate andthe organic phase was dried over sodium sulfate and concentrated. Theresidue was purified by silica gel column chromatography eluting with2:1 hexane:ethyl acetate mixture. The product was obtained as a whitesolid (1.29 g, 61% yield). M.p. 58-60° C.

¹H NMR (DMSO-d₆) 11.95 (1H, br); 7.85 (4H, m); 3.55 (2H, t, J=7.2 Hz);2.17 (2H, t, J=7.2 Hz); 1.7-1.4 (4H, m); 1.22 (10H, m).

Example 6

6-(Ethylsulfonylamino)hexanoic acid

A solution of ethanesulfonyl chloride (3.9 ml, 41.1 mmol) in dioxane (10ml) was added to a solution of 6-aminohexanoic acid (2 g, 15.2 mmol) in1N NaOH (56 ml) and dioxane (10 ml), while stirring at 0-5° C. The pH ofthe reaction mixture was adjusted to 8-9 by addition of 25% sodiumhydroxide solution. The mixture was allowed to warm to room temperatureand stirred for 30 minutes. Further 25% NaOH solution was added toadjust the pH to about 11. After 3.5 h 1N hydrochloric acid (15 ml) andethyl acetate (60 ml) were added. The organic layer was dried oversodium sulfate and concentrated. The residue was triturated with amixture of diethyl ether (5 ml) and hexane (15 ml). The solid wascollected by filtration and dried affording 1.3 g of the title compound(40% yield). ¹H NMR (DMSO-d₆): 11.9 (1H, s); 6.97 (1H, t, J=5.7 Hz);2.97 (2H, q, J=7.1); 2.88 (2H, q, J=6.6); 2.2 (2H, t, J=7.3); 1.47 (4H,m); 1.29 (2H, m); 1.18 (3H, t, J=7.3).

Example 7

8-(Ethylsulfonylamino)octanoic acid

A solution of ethanesulfonyl chloride (1.5 ml, 15.7 mmol) in dioxane (5ml) was added to a solution of 8-aminooctanoic acid (1 g, 6.28 mmol) in1N NaOH (22 ml) and dioxane (5 ml), while stirring at 0-5° C. Themixture was allowed to warm to room temperature and stirred for 3.5minutes. During this period, at 1 hour intervals, the pH was adjusted to7-8 by addition of 25% NaOH solution. The reaction mixture was washedwith diethyl ether (30 ml). The pH was adjusted to 1-2 by addition of 1NHCl and the mixture was extracted with ethyl acetate (70 ml). Theorganic layer was dried over sodium sulfate and concentrated. Theresidue was triturated with a mixture of diethyl ether. The solid wascollected by filtration and dried under vacuum affording 600 mg of thetitle compound (38% yield). ¹H NMR (DMSO-d₆): 11.9 (1H, s); 6.96 (1H, t,J=6 Hz); 2.96 (2H, q, J=7.1); 2.88 (2H, q, J=6.6); 2.2 (2H, t, J=7.3);1.45 (4H, m); 1.26 (6H, m); 1.18 (3H, t, J=7.3).

Example 10

10-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-decanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-;

Coupling by IBCF Method

To a solution of 10-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-decanoic acid(353 mg, 1.11 mmol), prepared according to Example 5 in anhydrousdichloromethane (10 ml), N-methylmorpholine was added (122 μl, 1.11mmol). The mixture was cooled to −15° C., then isobutyl chloroformate(144 μl, 1.11 mmol) was slowly added. After 15 minutes(2S)-2-amino-5-[[imino(nitroamino)methyl]amino]pentanamide, N-[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]-hydrochloridesalt of Example 3 (508 mg, 1.01 mmol) and further N-methylmorpholine(122 μl, 1.11 mmol) were added. The reaction mixture was stirred at−15-10° C. for 4 h, then concentrated to small volume and partitionedbetween ethyl acetate (20 ml) and water (10 ml). The aqueous phase wasfurther extracted with ethyl acetate (10 ml). The combined organicphases were dried over sodium sulfate and concentrated. The residue wastaken up with ethyl acetate (3 ml) and the solution was dropwise addedto hexane (120 ml) while stirring at room temperature. The solid wascollected by decantation and dried under vacuum (730 mg, 94%).

¹H NMR (DMSO-d₆): 8.81 (1H, d, J=2.7 Hz); 8.52 (1H, br); 7.98 (1H, d,J=8.05); 7.88 (2H, br); 7.85 (4H, m); 4.34 (1H, m); 4.06 (1H, dd,J=7.1); 3.56 (2H, t, J=7.14); 3.14 (2H,m); 2.55 (1H, m); 2.19 (1H, m);2.10 (2H, t, J=7.14); 2.0 (1H, m); 1.82 (1H, t, J=5.7); 1.78 (1H, m);1.73-1.35 (10H, m); 1.31 (1H, d, J=9.9); 1.24 (19H, m); 0.84 (9H, m);0.79 (3H, s).

Example 11

12-[(1,1-dimethylethoxy)carbonylamino]dodecanamide, N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]earbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-

The title compound was prepared according to the procedure for Example10 above from 12-tert-butoxycarbonylamino-dodecanoic acid and theproduct of Example 3, using appropriate reagents and reactionconditions. Analytical Data:

¹H NMR (DMSO-d6) 8.81 (1H, d, J=2.4); 8.52 (1H, br); 7.98 (1H, d,J=8.05); 7.85 (2H,v. br); 6.73 (1H, t, J=5.3); 4.33 (1H, m); 4.07 (1H,d, J=8.4); 3.14 (2H, m); 2.88 (2H, q, J=6.6); 2.56 (1H, m); 2.19 (1H,m); 2.10 (2H, t, J=7.1); 2.01 (1H, m); 1.83 (1H, t, J=.57); 1.78 (1H,m); 1.73-1.41 (8H, m); 1.36 (9H, s); 1.33-1.15 (25H, m); 0.84 (6H, d,J=6.5); 0.80 (3H, s).

Example 12

4-(methoxycarbonyl)heptanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]

The title compound was prepared according to the procedure for Example10 above from octanedioic acid monomethyl ester and the product ofExample 3, using appropriate reagents and reaction conditions.Analytical Data:

¹H -NMR (DMSO-d6): 8.80 (1H, br s); 8.51 (1H, br); 7.98 (1H, d, J=8.0Hz); 8.3-7.5 (2H, br); 4.32 (1H, m); 4.06 (1H, br d, J=8.4); 3.12 (2H,m); 2.55 (1H, m); 2.26 (2H, t, J=7.3); 2.18 (1H, m); 2.09 (2H, t,J=7.1); 2.01 (1H, m); 1.85-1.2 (19H, m); 1.23 (3H, s); 1.21 (3H, s);0.83 (6H, d, J=6.6); 0.79 (3H, s).

Example 13

11-Cyanoundecanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-.

PS-Carbodiimide (N-cyclohexylcarbodiimide-N′-propyloxymethylpolystyrene, 769 mg, 1 mmol, loading 1.31 mmol/g) and HOAt(1-Hydroxy-7-azabenzotriazole, 115 mg, 0.85 mmol) were added to asolution of 11-cyanoundecanoic acid (115 mg, 0.54 mmol) indichloromethane (DCM) (9 mL). After stirring for 10 minutes(2S)-2-amino-5-[[imino(nitroamino)methyl]amino]pentanamide,N-[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]-,hydrochloride salt of Example 3 (251 mg, 0.50 mmol) and DIPEA (0.128 ml,0.75 mmol) were added. The suspension was shaken overnight at roomtemperature and then the PS-Carbodiimide was filtered off and washedseveral times with DCM (4×6 mL). The organic phase was passed through aVARIAN CHEM ELUT cartridge for liquid-liquid extraction pre-conditionedwith saturated aqueous NaHCO₃ and finally washed with DCM (15 mL). Thesolvent was evaporated and the crude reaction was purified withnormal-phase ISOLUTE SPE-SI column (DCM 9, MeOH 1) to afford 200 mg ofthe desired compound (yield 61%). NMR (CDCl₃): 7.53 (s, br, 2H); 7.36(d, br, J=4.7 Hz, 1H); 6.88 (d, J=8.2 Hz, 1H); 4.46 (m, 1H); 4.15 (dd,J=8.5, 1.9 Hz, 1H); 3.19 (m, 2H); 2.93 (m, 1H); 2.23 (t, J=7.2 Hz, 2H);2.21 (m, 1H); 2.09 (t, J=7.5, 2H); 2.04 (m, 1H); 1.88 (t, J=5.4 Hz, 1H);1.77 (m, 1H); 1.69 (m, 1H); 1.64-1.43 (m, 9H); 1.40-1.26 (m, 4H); 1.26(s, 3H); 1.24-1.12 (m, 16H); 0.80 (d, J=6.6, 3H); 0.79 (d, J=6.6, 3H);0.73 (s, 3H). LC-MS 659.7, MH+. ESI POS; AQA; spray 4 kV/skimmer:20V/probe 250° C.

Example 14

9-Cyanononamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]

The title compound was prepared according to the procedure for Example13 above from 9-cyano-nonanoic acid and the product of Example 3, usingappropriate reagents and reaction conditions. Analytical Data: MS:MH+632.5

Example 15

6-(Acetylamino)hexanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]

The title compound was prepared according to the procedure for Example13 above from 6-acetylamino-hexanoic acid and the product of Example 3,using appropriate reagents and reaction conditions. Analytical Data: MS:[MH]+622.3

Example 16

12-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-dodecanamide,N-t(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]

The title compound was prepared according to the procedure for Example13 above from 11-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-undecanoic acidand the product of Example 3, using appropriate reagents and reactionconditions. Analytical Data: MS: [MH]+794.42

Example 17

6-(Ethanesulfonylamino)hexanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]

To a solution of 6-(ethylsulfonylamino)hexanoic acid (90 mg, 0.40 mmol,1.2 eq.), of Example 6, in dry DMF (10 ml) TBTU((N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate;130 mg, 0.40 mmol, 1.2 eq.) was added. The mixture was cooled to 0°-5°C. with an ice bath and NMM (0.12 ml, 1.08 mmol, 2.7 eq.) was added.After a few minutes(2S)-2-amino-5-[[imino(nitroamino)methyl]-amino]pentanamide,N-[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]-hydrochloridesalt (170 mg, 0.33 mmol, 1 eq.), of Example 3, was added. The mixturewas stirred at r.t. for 2 h, poured in water (150 ml) and extracted withethyl acetate (2×50 ml). The organic layer was washed with a solution ofcitric acid 2% (20 ml), sodium bicarbonate 2% (20 ml), and NaCl 2% (20ml), dried over sodium sulfate anhydrous and evaporated at reducedpressure. The residue was purified by column chromatography (silica gel,eluent ethyl acetate) to give 30 mg of amorphous solid (13% yield).

Analytical Data: ¹H-NMR (DMSO-d₆): 8.83 (1H, d, J=2.7 Hz); 8.51 (1H,br); 8.00 (1H, d, J=8.0 Hz); 8.3-7.5 (2H, br); 6.94 (1H, t, J=5.8): 4.32(1H, m); 4.06 (1H, dd, J=1.8, 8.6); 3.13 (2H, m); 2.95 (2H, q, J=7.3);2.87 (2H, q, J=6.7); 2.55 (1H, m); 2.19 (1H, m); 2.10 (2H, t, J=7.5);2.00 (1H, m); 1.85-1.1 (17H, m); 1.24 (3H, s); 1.21 (3H, s); 1.16 (3H,t, J=7.5); 0.83 (6H, d, J=6.6); 0.79 (3H, s).

Example 18

6-Benzenesulfonylaminohexanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]

The title compound was prepared according to the procedure for Example17 above from 8-phenylsulfonylamino-hexanoic acid and the product ofExample 3, using appropriate reagents and reaction conditions.Analytical Data: ¹H-NMR (DMSO-d₆): 8.83 (1H, d, J=2.8 Hz); 8.51 (1H,br); 7.97 (1H, d, J=7.8 Hz); 8.2-7.6 (2H, br); 7.77 (2H, m); 7.65-7.5(4H, m); 4.31 (1H, m); 4.05 (1H, dd, J=1.8, 8.6); 3.12 (2H, m); 2.69(2H, q, J=7.0); 2.54 (1H, m); 2.20 (1H, m); 2.05 (2H, t, J=7.5); 2.01(1H, m); 1.85-1.1 (21H, m); 1.22 (3H, s); 1.21 (3H, s); 0.82 (6H, d,J=6.6); 0.79 (3H, s).

Example 19

8-(Ethanesulfonylamino)octanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl

The title compound was prepared according to the procedure for Example17 above from 8-ethanesulfonylamino-octanoic acid (Example 7) and theproduct of Example 3, using appropriate reagents and reactionconditions. Analytical Data: ¹H-NMR (DMSO-d₆): 8.81 (1H, br s); 8.51(1H, br); 7.98 (1H, d, J=7.8 Hz); 8.3-7.5 (2H, br); 6.93 (1H, t, J=5.7):4.32 (1H, m); 4.06 (1H, dd, J=1.8, 8.6); 3.13 (2H, m); 2.95 (2H, q,J=7.3); 2.87 (2H, q, J=6.7); 2.55 (1H, m); 2.19 (1H, m); 2.10 (2H, t,J=7.0); 2.00 (1H, m); 1.85-1.1 (17H, m); 1.23 (3H, s); 1.21 (3H, s);1.16 (3H, t, J=7.3); 0.83 (6H, d, J=6.6); 0.79 (3H, s).

Example 20

Boronic acid,[(1R)-1-[[(2S)-5-[[imino(nitroamino)methyl]amino]-2-[(11-cianoundecanoyl)amino]-1-oxopentyl]amino]-3-methylbutyl]

To a solution of DMAP (4-Dimethylaminopyridine, 22.7 mg, 0.185 mmol) and11-cyanoundecanoic acid (97.2 mg, 0.46 mmol) in dichloromethane (5.2ml), PS-HOBT (1-Hydroxybenzotriazole-6-sulfonamidomethyl polystyrene,277 mg, 0.31 mmol, loading 1.12 mmol/g) was added. The mixture wasstirred at room temperature for 10 minutes, then a solution ofdiisopropylcarbodiimide (0.218 ml, 1.39 mmol) in DCM (dichloromethane,0.6 ml) was added. The suspension was shaken for 4 hours at roomtemperature and then the resin was filtered under nitrogen and washedseveral times with DMF (3×5 ml), DCM (3×5 ml), DMF (3×5 ml) and THF (3×5ml). The well dried resin was suspended in a solution of[(1R)-1-[[(2S)-2-amino-5-[[imino(nitroamino)-methyl]amino]-1-oxopentyl]amino]-3-methylbutyl]-boronicacid hydrochloride salt of Example 4 (55.2 mg, 0.15 mmol) and DIPEA(N,N-diisopropylethylamine, 0.051 ml, 0.293 mmol) in DCM (4 ml) and DMF(0.6 ml). The reaction mixture was shaken overnight at room temperature.The resin was filtered off and washed with DMF (10 ml) and DCM (2 ml)and the solvent was concentrated to dryness. The residue was purified byelution on a ISOLUTE SPE-DIOL cartridge, using DCM/Methanol mixturesfrom 95/5 to 50/50. The fractions containing the product were collectedand concentrated. The final purification was performed by elution on a 2g ISOLUTE SPE-SI normal phase cartridge using DCM/Methanol mixtures from100/0 to 50/50 (25 mg, 35% yield).

Analytical Data: MS: [M−18]H+508.5

Example 21

Boronic acid,[(1R)-1-[[(2S)-5-[[imino(nitroamino)methyl]amino]-2-[(9-cyanononanoyl)amino]-1-oxopentyl]amino]-3-methylbutyl]

The title compound was prepared according to the procedure for Example20 above from 11-cyano-nonanoic acid and the product of Example 4, usingappropriate reagents and reaction conditions. Analytical Data: MS:[M−18]H+480.1

Example 22

Boronic acid,[(1R)-1-[[(2S)-5-[[imino(nitroamino)methyl]amino]-2-[[7-(methoxycarbonyl)heptanoyl]amino]-1-oxopentyl]amino]-3-methylbutyl]

A mixture of 4-(methoxycarbonyl)heptanamide,N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-,prepared by the method of Example 12, (300 mg, 0.47 mmol),2-methylpropylboronic acid (96 mg, 0.94 mmol) and 4N hydrogen chloridedioxane solution (120 μl) in a 40:60 heterogeneous mixture ofmethanol:hexane (10 ml) was stirred at room temperature for 2 hours.Hexane (5 ml) was added, the mixture was stirred for a while, then thehexane layer was removed. Fresh hexane (5 ml), 4N hydrogen chloridedioxane solution (120 gl) and 2-methylpropylboronic acid (96 mg, 0.94mmol) were added and the mixture was stirred at room temperature for 2hours. The hexane layer was removed and the methanol phase was washedwith hexane (2×5 ml). The residue obtained upon concentration of themethanol phase was purified by silica gel column chromatography elutingwith a 40:40:20 acetone:methanol:hexane mixture. The product wasredissolved in ethyl acetate (200 ml) and the organic phase was washedwith water (2×10 ml), dried over sodium sulfate and concentrated. Theresidue was dissolved in the minimum amount of methanol, the solutionwas filtered through a cotton plug and concentrated. The residue wastriturated with diethyl ether. The solid was collected by decantation,then triturated with ethyl acetate (15 ml). The solid was collected bydecantation and dried under vacuum affording the product as a whitesolid (30 mg, 13% yield).

Analytical Data: ¹H NMR (DMSO-d₆): 8.60 (1H, d, J=8.4 Hz); 8.50 (1H,br); 8.06 (1H, d, J=7.9); 7.92 (2H, br); 4.36 (1H, m); 3.58 (3H, s);3.13 (2H, m); 2.55 (1H, m); 2.28 (2H, t, J=7.5); 2.12 (2H, m); 1.69 (1H,m); 1.49 (7H, m); 1.24(7H, m); 0.81 (6H, m). El. Anal. Calculated: C47.82% H 7.83% N 16.73% Found C 48.13% H 7.50% N 16.34%

Example 23

Boronic acid,[(1R)-1-[[(2S)-5-[[imino(nitroamino)methyl]amino]-2-[(10-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-1-oxodecyl]-)amino]-1-oxopentyl]amino]-3-methylbutyl]

The title compound was prepared according to the procedure for Example22 above from the product of Example 10, using appropriate reagents andreaction conditions. Analytical Data: ¹H-NMR (MeOH-d₄): 7.82 (4H, m);4.52 (1H, m); 3.66 (2H, t, J=7.3); 3.27 (2H, m); 2.75 (1H, m); 2.24 (2H,t, J=7.3 Hz); 1.9-1.2 (20H, m); 0.91 (6H, d, J=6.6).

Example 24

Boronic acid,[(1R)-1-[[(2S)-5-[[imino(nitroamino)methyl]amino]-2-[[6-(acetylamino)hexanoyl]amino]-1-oxopentyl]amino]-3-methylbutyl]

6-(Acetylamino)hexanamide, N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]-carbonyl]-4-[[imino(nitroamino)methyl]amino]butyl]-,of Example 15 (48 mg, 0.077 mmol), was dissolved in Et₂O (2 ml) and HCl37% (1 ml) was added carefully at 0° C. The reaction mixture was allowedto warm to room temperature and to shake overnight. The mixture wasconcentrated to dryness and the residue was dissolved in MeOH (1 ml) andeluted through an ISOLUTE PSA cartridge with MeOH (10 ml). The solventwas evaporated and the crude product was purified eluting through a 500mg ISOLUTE SPE-SI cartridge using DCM/Methanol mixtures from 90/10 to50/50 to afford the title compound (19.4 mg, 52% yield).

Analytical Data: MS: [M−18]H+470.2

Utility

Compound Activity

The present compounds can inhibit proteasome activity. Table F-1 belowprovides data related to several example compounds of the invention withrespect to, for example, ability to inhibit proteasome activity.

Methods and Compositions

Compounds of the present invention can inhibit the activity ofproteasome leading to the inhibition or blocking of a variety ofintracellular functions with which the proteasome is directly orindirectly associated. For example, proteasome inhibitors can modulate,such as induce, apoptosis in a cell. In some embodiments, the compoundsherein can kill tumor cells by induction of apoptosis. Thus, the presentcompounds can be used to treat cancer, tumors or other proliferativedisorders.

In further embodiments, inhibition of proteasome function by compoundsof the invention can inhibit the activation or processing oftranscription factor NF-κB. This protein plays a role in the regulationof genes involved in the immune and inflammatory responses as well as incell viability. Inhibition of proteasome function can also inhibit theubiquitination/proteolysis pathway. This pathway catalyzes, inter alia,selective degradation of highly abnormal proteins and short-livedregulatory proteins. In some embodiments, compounds of the invention canprevent the degradation of p53 which is typically degraded by theubiquitin-dependent pathway. The ubiquitination/proteolysis pathway alsois involved in the processing of internalized cellular or viral antigensinto antigenic peptides that bind to MHC-I molecules. Thus, thecompounds of the invention can be used to reduce the activity of thecytosolic ATP-ubiquitin-dependent proteolytic system in a number of celltypes.

Accordingly, the usefulness of such compounds can include therapeutics,such as the treatment of various diseases or disorders associated withproteasome. The methods include administering a therapeuticallyeffective amount of a compound of the invention, or composition thereof,to a mammal, such as a human having a disease or disorder associatedwith proteasome. The phrase “therapeutically effective amount” refers toan amount sufficient to prevent, alleviate, or ameliorate anyphenomenon, such as a cause or symptom, known in the art to beassociated with the disease or disorder.

Treatable diseases or disorders (abnormal physical conditions) can beassociated with either normal or abnormal activities of proteasome, suchas the regulation of apoptosis. Numerous diseases or disorders that areassociated with proteasome, or that are desirably treated by inductionof apoptosis, are known and include, for example, various cancers andtumors including those associated with skin, prostate, colorectal,pancreas, kidney, ovary, mammary, liver, tongue, lung, and smooth muscletissues. Preferred tumors that can be treated with proteasome inhibitorsinclude, but are not limited to hematological tumors, such as, forexample, leukemias, lymphomas, non-Hodgkin lymphoma, myeloma, multiplemyeloma, as well as solid tumors such as, for example, colorectal,mammary, prostate, lung, and pancreas tumors. In order to elicittherapeutic effects, the proteasome inhibitors can be administered topatients as single agents or in combination with one or more antitumoror anticancer agent and/or radiotherapy. Examples of other anti-tumor oranti-cancer agents which can be advantageously administeredconcomitantly with a proteasome inhibitor include but are not limitedto, adriamycin, daunomycin, methotrexate, vincristin, 6-mercaptopurine,cytosine arabinoside, cyclophosphamide, 5-FU, hexamethylmelamine,carboplatin, cisplatin, idarubycin, paclitaxel, docetaxel, topotecan,irinotecam, gemcitabine, L-PAM, BCNU and VP-16. Methods for determiningapoptosis in vitro are well known in the art and kits are availablecommercially. See for example the Apo-ONE™ Homogeneous Caspase-3/7 Assayfrom Promega Corporation, Madison Wis., USA (Technical Bulletin No.295,revised February 2002, Promega Corporation).

Further diseases or disorders associated with the proteasome includeaccelerated or enhanced proteolysis that occurs in atrophying muscles,such as is often associated with activation of a nonlysomalATP-requiring process involving ubiquitin. Accelerated or enhancedproteolysis can be the result of any of numerous causes includingsepsis, burns, trauma, cancer, infection, neurodegenerative diseasessuch as muscular dystrophy, acidosis, or spinal/nerve injuries,corticosteroid use, fever, stress, and starvation. Compounds of theinvention can be tested for inhibition of muscle wastage by any variousprocedures known in the art such as by measuring urinary excretion ofmodified amino acid 3-methylhistidine (see, e.g., Young, et al.,Federation Proc., 1978, 37, 229).

Compounds of the present invention can be further used to treat orprevent diseases or disorders associated with activity of NF-κBincluding for example, human immunodeficiency virus (HIV) infection andinflammatory disorders resulting from, for example, transplantationrejection, arthritis, infection, inflammatory bowel disease, asthma,osteoporosis, osteoarthritis, psoriasis, restenosis, and autoimmunediseases. Accordingly, a process that prevents activation of NF-κB inpatients suffering from such a disease would be therapeuticallybeneficial. Inhibition of the NF-κB activity can be measured by using aDNA binding assay such a described in Palombella, et al., Cell, 1994,78, 773.

Those of ordinary skill in the art can readily identify individuals whoare prone to or suspected of suffering from such diseases or disordersusing standard diagnostic techniques.

Example A

Assay for Chymotrypsin-like Activity of 20S Human Erythrocyte Proteasome(HEP)

Proteasome chymotrypsin-like activity of compounds of the invention wasassayed according to the following procedure.

In 96-well microtiter plates, 20S Human Erythrocyte Proteasome (HEP),purchased from Immatics Biotechnologies Inc., Tubingen, Germany wasplated at 0.2 μg/mL (about 0.6 nM catalytic sites) in 0.04% SDS 20 mMTris buffer. A fluorimetric substrate Suc-LLVY-AMC(succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin), purchased fromSigma Inc., St. Louis, Mo., USA was added to a final concentration of100 μM from a stock solution of 10 mM in dimethylsulfoxide. Reactionvolumes were 100 μl per well. After incubation for various periods oftime at 37° C., the concentration of free AMC (aminomethylcoumarin) wasdetermined on a Perkin Elmer HTS 7000 Plus microplate reader, excitation370 nM and emission 465 nM. Proteasome activity was determined underconditions in which substrate hydrolysis increased linearly with timeand the change in fluorescence signal was proportional to theconcentration of free AMC.

Example B

Assay for Activity of α-Chymotrypsin

In 96-well microtiter plates bovine α-chymotrypsin, purchased from SigmaInc., was plated at 10 ng/mL (about 2 pM catalytic sites) in 0.5 M NaCl50 mM Hepes buffer. A fluorimetric substrate Suc-AAPF-AMC(succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin), purchased fromSigma Inc., St. Louis, Mo., USA was added to a final concentration of 25μM from a stock solution of 10 mM in dimethylsulfoxide. Reaction volumeswere 100 μl per well. After incubation for various periods of time atroom temperature, the concentration of free AMC was determined on aPerkin Elmer HTS 7000 Plus microplate reader, excitation 370 nM andemission 465 nM. α-Chymotrypsin activity was determined under conditionsin which substrate hydrolysis increased linearly with time and thechange in fluorescence signal was proportional to the concentration offree AMC.

Example C

Determination of IC₅₀ Values for HEP and α-Chymotrypsin Inhibitors

IC₅₀ values are typically defined as the concentration of a compoundnecessary to produce 50% inhibition of the enzyme's activity. IC₅₀values are useful indicators of the activity of a compound for itsdesignated use. The proteasome inhibitors of the invention can beconsidered active if they have IC₅₀ values of less than about 1micromolar for inhibition of human erythrocyte proteasome (HEP). In someembodiments, the inhibitors show some specificity for HEP and the ratioof the IC₅₀ for inhibition of bovine α-chymotrypsin versus the IC₅₀ forinhibition of HEP, i.e, IC₅₀ (α-Chymotripsin)/IC₅₀ (HEP), is greaterthen about 100.

Inhibition of the chymotrypsin-like activity of HEP and of bovineα-chymotrypsin was determined by incubating the enzyme with variousconcentrations of putative inhibitors for 15 minutes at 37° C. (or roomtemperature for α-chymotrypsin) prior to the addition of substrate. Eachexperimental condition was evaluated in triplicate, and replicateexperiments were performed for the inhibitors described herein.

Compounds of the present invention are considered active in the aboveidentified assay if their IC₅₀ values for inhibition of HEP are lessthan 1000 nanoMolar. Preferably compounds of the present invention willhave IC₅₀ values for inhibition of HEP less than 100 nanoMolar. Morepreferably compounds of the present invention will have IC₅₀ values forinhibition of HEP less than 10 nanoMolar. Compounds of the presentinvention have demonstrated, in the above identified assay, IC₅₀ valuesfor inhibition of HEP less than 1000 nanoMolar.

Example D

Cellular Assay for Chymotrypsin-like activity of Proteasome in Molt-4Cell Line

The chymotrypsin-like activity of proteasome in Molt-4 cells (humanleukemia) was assayed according to the following procedure. A briefdescription of the method was published previously (Harding et al., J.Immunol., 1995, 155, 1767).

Molt-4 cells were washed and resuspended in HEPES-buffered Saline (5.4mM KCl, 120 mM NaCl, 25 mM Glucose, 1.5 mM MgSO₄, 1 mM Na pyruvate, 20mM Hepes) and plated in 96-well microtiter white plates to a finalconcentration of 6×10⁶ cells/mL. Then various 5× proteasome inhibitorconcentrations (or diluted DMSO for controls), prepared from 250×DMSOsolutions by diluting 50-fold using HEPES-buffered saline, were added tothe plate to a final 1× concentration. After 15 minutes incubation at37° C., a fluorimetric cell permeable substrate (MeOSuc-FLF-AFC)(methoxysuccinyl-Phe-Leu-Phe-7-amido-4-trifluoromethylcoumarin)purchased from Enzyme Systems Products, catalogue number AFC-88, wasadded to each well at a final concentration of 25 μM from a stocksolution of 20 mM in DMSO. Reaction volumes were 100 μl per well.

The concentration of free AFC was monitored every 1.5 min for 30 min (22cycles) on a Polastar Optima, BMG Labtechnologies microplate reader,using an excitation wavelength of 390 nm and emission wavelength of 520nm. Proteasome activity was determined under conditions in whichsubstrate hydrolysis increased linearly with time and the change influorescent signal was proportional to the concentration of free AFC.

Example E

Determination of EC₅₀ values for proteasome inhibitors in MOLT-4 CellLine

EC₅₀ values are typically defined as the concentration of a compoundrequired to produce an inhibition of the enzyme's activity halfwaybetween the minimum and the maximum response (0% and 85-90% respectivelyfor this assay). EC₅₀ values are useful indicators of the activity of acompound for its designated use. The compounds of the invention can beconsidered active if they have an EC₅₀ of less than about 10 micromolar.

Inhibition of chymotrypsin-like activity of proteasome in Molt-4 cellswas determined by incubating cells with various concentrations ofputative inhibitors for 15 minutes at 37° C. prior to the addition ofsubstrate. Each experimental condition was evaluated in triplicate, andreplicate experiments were performed for the inhibitors describedherein.

Compounds of the present invention are considered active in the aboveidentified assay if their EC₅₀ values for proteasome inhibition inMOLT-4 are less than 10 microMolar. Preferably compounds of the presentinvention will have EC₅₀ values for proteasome inhibition in MOLT-4 lessthan 2 microMolar. More preferably compounds of the present inventionwill have EC₅₀ values for proteasome inhibition in MOLT-4 less than 200nanomolar. Compounds of the present invention have demonstrated, in theabove identified assay, EC₅₀ values for proteasome inhibition in MOLT-4cells of less than 10 microMolar.

Example F

Assay for Trypsin-like Activity of the Proteasome

The trypsin-like activity of human proteasome can be assayed asdescribed above with the following modifications. Reactions can becarried out in Tris-glycerol buffer (pH 9.5) supplemented with 1 mM2-mercaptoethanol, and the substrate can be a fluorogenic substrate suchas benzyloxycarbonyl--Phe--Arg--AMC (100 μM).

After incubation for various periods of time at 37° C., theconcentration of free AMC can be determined on a Fluoroskan IIspectrofluorimeter with an excitation filter of 390 nm and an emissionfilter of 460 nm. Protease activity can be determined under conditionsin which substrate hydrolysis increases linearly with time and thechange in fluorescence is proportional to the concentration of free AMC.

Example G

In vivo Inhibition of Cellular Muscle Breakdown

The effect of inhibitors on the unweighting atrophy of the soleus musclein juvenile rats can be determined by, for example, the proceduresdescribed in Tischler, Metabolism, 1990, 39, 756. For example, juvenilefemale Sprague-Dawley rats (80-90 g) can be tail-cast, hind limbsuspended as described in Jaspers, et al., J. Appl. Physiol., 1984, 57,1472. The animal's hind limbs can be elevated above the floor of thecage with each animal housed individually. Animals can have free accessto food and water, and can be weighed at the time of suspension and attime of termination. During the suspension period the animals can bechecked daily to ensure that their toes are not touching the floor ofthe cage, and that there is no swelling of the tail due to the cast.

Experimental Design—Part 1

Each experiment can begin with the suspension of 20 rats which arerandomly divided into 4 groups of 5 animals each. Group A can besuspended for 2 days, providing baseline data to approximate the soleusmuscle size in other animals suspended for longer times. Average bodyweights for the groups at the outset of the study can be compared andused as a correction factor for body size differences. Group B can be asecond control group which has the soleus of one limb treated with anaqueous solution of mersalyl after two days of unweighting, todemonstrate the ability to slow muscle atrophy during unweighting, foreach group of animals. At 2 days after unweighting commences, an aqueoussolution of mersalyl (200 nM; 4 .mu.l/100 g initial body wt) can beinjected into one soleus. The contralateral muscle can be injected witha similar volume of 0.9% saline (“Vehicle”). The animals can bemaintained under Innovar-vet (10 μl/100 g body wt) tranquilizationduring the in situ injection procedure. After the injections, theanimals can be suspended for an additional 24 hours and the soleus canbe removed. Groups C and D for each experiment can be used for testingeach of two different embodiments of the disclosed compounds. Animalscan be treated as in group B, except that 1 mM proteasome inhibitor,contained in dimethysulfoxide (DMSO), can be injected into the soleus ofone leg and DMSO only into the contralateral soleus. Thus eachexperiment consists of two control groups and the testing of proteasomeinhibitors of the invention. The completion of five such experimentswith different pairs of inhibitors provides for an “n” value of 10 fortesting each inhibitor and each can be tested in two different shipmentsof animals.

Processing of the Soleus Muscle—Part 1

After the animal is sacrificed, the soleus can be excised, trimmed offat and connective tissue, and carefully weighed. The muscle can thenhomogenized in 10% trichloroacetic acid (TCA) and the precipitatedprotein pelleted by centrifugation. The pellet can then be washed oncewith 10% TCA and once with ethanol:ether (1:1). The final pellet can besolubilized in 4 ml of 1N sodium hydroxide. The sample can be thenanalyzed for protein content by the biuret procedure, using albumin as astandard.

Data Analysis—Part 1

The effect of inhibitors on total muscle protein content can be examinedprimarily by paired comparison with the untreated contralateral muscle.The ratio of contents can be calculated and then analyzed statisticallyby analysis of variance (“ANOVA”). The left leg can always be thetreated leg so that the protein content ratios can be compared to thenon-treated control animals as well. In this way, a significantdifference can be shown by comparing the protein content of the twolegs, as well as the relative effectiveness of the tested inhibitors. Apaired student test can also be performed for the effect of eachseparate treatment. The non-treated control data also provide anestimate of protein content of day 2. This allows approximation of theprotein changes over the 24 hours of treatment for each of the Groups B,C, and D.

Experimental Design—Part 2

Each experiment can consist of 10 animals with groups of 5 animals beingtested with one of the inhibitors for its effect on protein synthesis.Control animals are not needed for this aspect of the study as thecontralateral DMSO-treated muscle serves as the paired control for theinhibitor-treated muscle. Each group can be injected as described forgroups C and D in part 1. Twenty-four hours after the in situ treatmentthe fractional rate of protein synthesis can be analyzed in both soleusmuscles. Each muscle can be injected with a 0.9% saline solution (3.5μl/100 g final body wt) containing ³H-phenylalanine (50 mM; 1μCi/^(m)l). Fifteen minutes later the middle two-thirds of the musclecan be excised and the muscle can be processed as described below.

Processing of the Soleus Muscle—Part 2

The muscle can be first washed for 10 minutes in 0.84% saline containing0.5 mM cycloheximide, to terminate protein synthesis, and 20 mMcycloleucine, to trap phenylalanine in the cell. The muscle can then behomogenized in 2.5 mL of ice-cold 2% perchloric acid. The precipitatedprotein can be pelleted by centrifugation. One aliquot of thesupernatant can be taken for liquid scintillation counting and anotheraliquot can be processed for conversion of phenylalanine tophenethylamine to determine the soluble phenylalanine concentrationfluorometrically. See, e.g., Garlick, et al., Biochem. J., 1980, 192,719. These values can provide the intracellular specific activity. Thespecific activity of phenylalanine in the muscle protein can bedetermined after hydrolyzing the protein by heating in 6N HCl. The aminoacids released can be solubilized in buffer. One aliquot can be takenfor scintillation counting and another for analysis of phenylalanine asfor the supernatant fraction. The fractional rate of protein synthesiscan be calculated as: protein specific activity/intracellular specificactivity.times.time.

Data Analysis—Part 2

Analyses of protein synthesis can be on a paired basis for eachinhibitor. Student paired t test comparisons of the contralateralmuscles can determine whether there is any effect of the inhibitor onprotein synthesis. Protein breakdown can be calculated approximately asthe fractional rate of protein synthesis (from part 2) plus thefractional rate of protein accretion (from part 1), where protein lossyields a negative value for protein accretion.

Qualitatively the ability of inhibitors to slow protein loss withoutaffecting protein synthesis indicates a slowing of protein degradation.

Example H

In vivo Investigation of Anti-Tumor Activity

Materials

The proteasome inhibitors used for in vivo studies can be formulated inan appropriate medium for intravenous (iv) or oral (po) administration.For example, for the iv administration the compounds can be administereddissolved in 0.9% NaCl, or in mixtures of 0.9% NaCl, solutol HS15 anddimethylsulfoxide, for example in the ratio 87:10:3 (v:v:v),respectively.

Cell Lines

The following human and murine tumor cell lines of differenthistological origine can be used to test the antitumor activity of thecompounds of the invention: H460 (human, lung), A2780 (human, ovary),PC-3 (human, prostate), LoVo (human, colon), HCT116 (human, colon),BXPC3 (human, pancreatic), PANC-1 (human, pancreatic), MX-1 (human,mammary), MOLT (human, leukemia), multiple myeloma (human, myeloma), YC8(murine, lymphoma), L1210 (murine, leukemia), 3LL (murine, lung).

Animal Species

5-6 weeks immunocompetent or immunodeprived mice are purchased fromcommercial sources, for example from Harlan (Correzzana, Mi Italy). CD1nu/nu mice are maintained under sterile conditions; sterilized cages,bedding, food and acidified water are used.

Tumor Cell Implantation and Growth

Solid tumor models of different hystotype (lung, ovary, breast,prostate, pancreatic, colon) can be transplanted subcutaneously (sc.)into the axillary region of immunocompetent mice (murine models) or inimmunodeprived mice (human models). Human tumor cell lines, originallyobtained from ATCC, can be adapted to grow “in vivo” as solid tumor from“in vitro culture”.

Hematological human or murine tumor models can be transplanted intodifferent sites (iv, ip , ic or sc) in immunocompetent mice (murinetumors) or in immunodeprived mice (human leukemia, lymphoma and myelomamodels), according to their highest tumor take.

Drug Treatment

Mice bearing solid (staged) or hematological tumors are randomized inexperimental groups (10 mice/group). For solid tumors, an average tumorweight of 80-100 mg for each group is considered to start the treatment;mice with the smallest and largest tumors are discarded.

Experimental groups are randomly assigned to the drug treatment and tothe control group. Animals can be treated iv or orally, depending on theoral bioavailability with the compounds following different treatmentschedules: iv weekly or twice weekly, or by daily oral administration.

On solid tumor models, drug treatment can begin when the tumor sizeranges between 80-100 mg after tumor transplantation (Day 0).

The compounds can be administered in a volume of 10 mL/Kg bodyweight/mouse in the appropriate solvent.

Parameters of Antitumor Activity

The following parameters can be assessed for the evaluation of theantitumor activity:

-   -   growth of primary solid tumor; in each mouse is monitored by        caliper measurement twice weekly;    -   survival time of treated mice as compared to control mice    -   twice weekly body weight evaluation of individual mice.

The tumor growth inhibition, TWI% (percentage of primary tumor growthinhibition in comparison with vehicle treated control groups) or theRelative tumor growth inhibition, RTWI% in case of staged tumors, isevaluated one week after the last drug treatment and the Tumor weight(TW) can be calculated as follows:TW=½ab²where a and b are long and short diameters of the tumor mass in mm.

The antitumor activity can be determined as tumor weight inhibition (TWI%), which is calculated according to the formula:${{TWI}\quad\%} = {100 - {\frac{{mean}\quad{TW}\quad{treated}}{{mean}\quad{TW}\quad{controls}} \times 100}}$

The RTWI% (relative percentage of primary tumor growth inhibition incomparison with vehicle treated control groups) is evaluated one weekafter the last drug treatment, according to the following formula:${{RTWI}\quad\%} = {100 - {\frac{{mean}\quad{RV}\quad{of}\quad{treated}\quad{mice}}{{mean}\quad{RV}\quad{of}\quad{controls}\quad{mice}} \times 100}}$where${RV} = \frac{{Vt}\quad\left( {{tumor}\quad{weight}\quad{on}\quad{day}\quad t} \right)}{{Vo}\quad\left( {{intitial}\quad{tumor}\quad{weight}\quad{at}\quad{the}\quad{outset}\quad{of}\quad{treatment}} \right)}$

The Percent of Tumor Regression can be calculated as regressions interms of relative tumor weight, determined as tumor weight at given daydivided by initial tumor weight at the outset the experiment.

On haematological tumour models the antitumor activity can be determinedas percentage increase of the median survival time of mice expressed asthe ratio (T/C%) of the median survival time of the treated group (T) tothat of the control group (C). Animals which are tumour-free at the endof the experiment (60 days after transplantation) are excluded from thecalculation and considered as long term survivors (LTS).

Evaluation of Toxicity in Tumor Bearing Mice

Toxicity can be evaluated daily on the basis of the gross autopsyfindings and the weight loss. Mice are considered to have died oftoxicity when death occurs before the death of vehicle treated controlanimals, or when significant body weight loss (>20%), and/or spleen andliver size reduction are observed.

The BWC% (Body weight change %) is assessed as follow: 100−(mean bodyweight of mice at given day/mean body weight at start of treatment)×100.This value is determined one week after the last treatment with the testcompound.

Example K

In vitro Viability of Cells

The IC₅₀ values measuring in vitro viability of cells in the presence oftest compounds can be determined according to the following procedure.Cells can be seeded in 96-well plates at varying densities and thenassayed using the Calcein-AM viability assay after 24 hours to determinethe optimal final density for each cell type. Cells can then be seededin 96-well plates at the determined density in 100 μL of an appropriatecell media known to one skilled in the art.

Serial dilutions of test compounds can be made so that theconcentrations are twice the desired concentration to be evaluated. When100 μL of the dilution is then added to the cells plated in 100 μL ofmedia, a final concentration of, for example, 0, 11.7, 46.9, 187.5, 375,and 750 nM can be obtained. Compounds can be added to the plates threeto four hours after seeding the cells, then the plates can be incubatedat 37° C. for the desired time point (e.g., one, two, or three days).

Calcein-AM viability assays can be conducted at the desired time pointsas follows. Media can be aspirated using a manifold and metal plate toleave approximately 50 EL/well. The wells can be washed three times with200 μL DPBS, aspirating each time with the manifold to leave 50 μL/well.A 8 μM solution of Calcein-AM in DPBS can be prepared and 150 μL can beadded to each well. The plates can then be incubated at 37° C. for 30minutes. After incubation, calcein can be aspirated with the manifoldand cells can be washed with 200 μL DPBS as before. After finalaspiration, fluorescence can be measured using a Cytofluor 2300fluorescence plate reader. Negative controls can contain media and nocells, and experiments can be conducted in triplicate.

Example L

Kinetic Experiments in vitro

Compounds of the invention can be tested for proteasorne inhibitoryactivity using a protocol described in Rock, et al., Cell, 1994, 78,761. According to this procedure, dissociation constants (K_(i)) for theequilibrium established when proteasome and test compound interact toform a complex. The reactions can be carried out using SDS-activated 20Sproteasome from rabbit muscle, and the proteasome substrate can beSuc-LLVY-AMC.

Example M

Inhibition of Activation of NF-κB

Compounds of the invention can be tested for inhibiting the activity ofNF-κB by carrying out the assay described in Palombella, et al., Cell,1994, 78, 773). For example, MG63 osteocarcinoma cells can be stimulatedby treatment with TNF-α for designated times. Whole cell extracts can beprepared and analyzed by electrophoretic mobility shift assays using thePRDII probe from the human IFN-β gene promoter.

Example N

Compound Activity

Using the assays of Example C and Example E above the following TableF-1 demonstrates the utility of compounds of the invention forproteasome inhibition. In the following Tables, for the inhibition ofHEP, Example C, compounds of the present invention with a “+” are lessthan 1000 nanoMolar; compounds of the present invention with a “++” areless than 100 nanoMolar; and compounds of the present invention with a“+++” are less than 10 nanoMolar in IC₅₀ for HEP inhibition. In thefollowing Tables, for the inhibition of MOLT4, Example E, compounds ofthe present invention with a “+” are less than 10000 nanoMolar;compounds of the present invention with a “++” are less than 2000nanoMolar; and compounds of the present invention with a “+++” are lessthan 200 nanoMolar in EC₅₀ for HEP inhibition. Where “>+” occursactivity was greater than the limits of the assay. Where no IC₅₀ valueor EC₅₀ value is represented, data has yet to be determined. TABLE F-1Example # HEP (IC₅₀) MOLT4 (EC₅₀) 10 +++ +++ 11 +++ +++ 12 +++ ++ 13 ++++++ 14 +++ +++ 15 ++ 16 +++ +++ 17 ++ >+ 18 +++ ++ 19 +++ +++ 20 +++ +21 +++ >+ 22 +++ >+ 23 +++ +++ 24 +++ >+Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of Formula (I) can beadministered in the form of pharmaceutical compositions. Thesecompositions can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal, and can be prepared in a manner well known in thepharmaceutical art.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of Formula (I)above in combination with one or more pharmaceutically acceptablecarriers. In making the compositions of the invention, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 100 mg, more usually about 10 to about30 mg, of the active ingredient. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is referred to as “therapeuticallyeffective amount.” Effective doses will depend on the disease conditionbeing treated as well as by the judgement of the attending cliniciandepending upon factors such as the severity of the disease, the age,weight and general condition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention canvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound, thehealth and condition of the patient, and the judgment of the prescribingphysician. The proportion or concentration of a compound of theinvention in a pharmaceutical composition can vary depending upon anumber of factors including dosage, chemical characteristics (e.g.,hydrophobicity), and the route of administration. For example, thecompounds of the invention can be provided in an aqueous physiologicalbuffer solution containing about 0.1 to about 10% w/v of the compoundfor parenteral adminstration. Some typical dose ranges are from about 1μg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. The dosage is likely to depend on such variables as the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, formulation of the excipient, and its route ofadministration. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of inflammatory diseases, whichcomprise one or more containers containing a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of Formula(I). Such kits can further include, if desired, one or more of variousconventional pharmaceutical kit components, such as, for example,containers with one or more pharmaceutically acceptable carriers,additional containers, etc., as will be readily apparent to thoseskilled in the art. Instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

1. A compound of Formula (I):

or a pharmaceutically acceptable salt, stereoisomeric or tautomeric formthereof, wherein: R¹ is C₁-C₄ alkyl; Y is or H, CN, NO₂, or a guanidinoprotecting group; Z is —CN, —C(═O)OR⁵, phthalimido, —NHSO₂R⁶, or —NR³R⁴;R³ is H or C₁-C₄ alkyl; R⁴ is selected from the group aryl-SO₂—,aryl-C(═O)—, aralkyl-C(═O)—, alkyl-C(═O)—, aryl-OC(═O)—,aralkyl-OC(═O)—, alkyl-OC(═O)—, aryl-NHC(═O)—, and alkyl-NHC(═O)—;wherein aryl or aralkyl is optionally substituted by one or more methylor methoxy groups; R₅ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,wherein R⁵ is optionally substituted by one or more halo, C₁-C₄ alkyl,aryl, or heteroaryl; R⁶ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,wherein R⁶ is optionally substituted by one or more halo, C₁-C₄ alkyl,aryl, or heteroaryl; Q is selected from B(OR⁹)₂, pinanediol boronicester, pinacol boronic ester, 1,2-ethanediol boronic ester,1,3-propanediol boronic ester, 1,2-propanediol boronic ester,2,3-butanediol boronic ester, 1,1,2,2-tetramethylethanediol boronicester, 1,2-diisopropylethanediol boronic ester, 5,6-decanediol boronicester, 1,2-dicyclohexylethanediol boronic ester, bicyclohexyl-1,1′-diolboronic ester, and 1,2-diphenyl-1,2-ethanediol boronic ester; R⁹ is H,C₁-C₄ alkyl, cycloalkyl, cycloalkylalkyl, aryl, or aralkyl; m is 2, 3,or 4; and n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or
 14. 2. Thecompound of claim 1 wherein R¹ is ethyl, propyl, or butyl.
 3. Thecompound of claim 1 wherein R¹ is 2-propyl.
 4. The compound of claim 1wherein Q is pinanediol boronic ester.
 5. The compound of claim 1wherein Q is bicyclohexyl-1,1′-diol boronic ester.
 6. The compound ofclaim 1 wherein Q is B(OH)₂.
 7. The compound of claim 1 wherein Z isphthalimido, NRR⁴, or CN.
 8. The compound of claim 1 wherein n is 8, 9,10, or
 11. 9. The compound of claim 1 wherein m is
 3. 10. The compoundof claim 1 wherein n is 8, 9, 1 0, or 11; and m is
 3. 11. A compound ofclaim 1 having Formula (I):

or a pharmaceutically acceptable salt, stereoisomeric or tautomeric formthereof, wherein: R¹ is 2-propyl; Y is —NO₂; Z is —CN, —C(═O)OR⁵,phthalimido, —NHSO₂R⁶, or —NHR⁴; R⁴ is selected from the grouparyl-SO₂—, alkyl-SO₂—, aryl-C(═O)—, aralkyl-C(═O)—, alkyl-C(═O)—,aryl-OC(═O)—, aralkyl-OC(═O)—, alkyl-OC(═O)—, aryl-NHC(═O)—, andalkyl-NHC(═O)—; wherein said aryl or aralkyl is optionally substitutedby one or more methyl or methoxy groups; R⁵ is H or C₁-C₆ alkyl, whereinsaid alkyl is optionally substituted with one or more halo, aryl, orheteroaryl; R⁶ is C₁-C₆ alkyl, wherein said alkyl is optionallysubstituted with one or more halo, aryl, or heteroaryl; Q is selectedfrom B(OR⁹)₂, pinanediol boronic ester, pinacol boronic ester,1,2-ethanediol boronic ester, 1,3-propanediol boronic ester,1,2-propanediol boronic ester, 2,3-butanediol boronic ester,1,1,2,2-tetramethylethanediol boronic ester, 1,2-diisopropylethanediolboronic ester, 5,6-decanediol boronic ester, 1,2-dicyclohexylethanediolboronic ester, bicyclohexyl-1,1′-diol boronic ester, and1,2-diphenyl-1,2-ethanediol boronic ester; R⁹ is H or C₁-C₄ alkyl; m is3; and n is 4, 5, 6, 7, 8, 9, 10 or
 11. 12. A compound of claim 1 ofFormula (I-s):

or a pharmaceutically acceptable salt, stereoisomeric or tautomeric formthereof, wherein: R¹ is C₁-C₄ alkyl; Y is CN or NO₂; Z is —CN,—C(═O)OR⁵, phthalimido, —NHSO₂R⁶, or —NR³R⁴; R³ is H or C₁-C₄ alkyl; R⁴is selected from the group aryl-SO₂—, alkyl-SO₂—, aryl-C(═O)—,aralkyl-C(═O)—, alkyl-C(═O)—, aryl-OC(═O)—, aralkyl-OC(═O)—,alkyl-OC(═O)—, aryl-NHC(═O)—, and alkyl-NHC(═O)—; wherein said aryl oraralkyl is optionally substituted by one or more methyl or methoxygroups; R⁵ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, wherein R⁵is optionally substituted by one or more halo, C₁-C₄ alkyl, aryl, orheteroaryl; R⁶ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, wherein R⁶is optionally substituted by one or more halo, C₁-C₄ alkyl, aryl, orheteroaryl; Q is selected from B(OR⁹)₂, pinanediol boronic ester,1,2-dicyclohexylethanediol boronic ester, and bicyclohexyl-1,1′-diolboronic ester; R⁹ is H or C₁-C₄ alkyl; m is 2, 3, or 4; and n is 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or
 14. 13. A compound of claim 1 havingthe formula:

or a pharmaceutically acceptable salt or free base form thereof.
 14. Acomposition comprising a compound of any one of claims 1-13 and apharmaceutically acceptable carrier.
 15. A method of inhibiting activityof proteasome comprising contacting a compound of any one of claims 1-13with said proteasome.
 16. A method of treating cancer comprisingadministering to a mammal having or predisposed to said cancer atherapeutically effective amount of a compound of any one of claims1-13.
 17. A method of treating cancer treating cancer comprisingadministering to a mammal having or predisposed to said cancer atherapeutically effective amount of a compound of any one of claims1-13, and wherein said cancer is selected from skin, prostate,colorectal, pancreas, kidney, ovary, mammary, liver, tongue, lung, andsmooth muscle tissue.
 18. A method of treating cancer comprisingadministering to a mammal having or predisposed to said cancer atherapeutically effective amount of a compound of any one of claims1-13, and wherein said cancer is selected from leukemia, lymphoma,non-Hodgkin lymphoma, myeloma, and multiple myeloma.
 19. A method oftreating cancer comprising administering to a mammal having orpredisposed to said cancer a therapeutically effective amount of acompound of any one of claims 1-13 in combination with one or moreantitumor or anticancer agent and/or radiotherapy.
 20. A method ofinhibiting activity of transcription factor NF-κB comprising contactingIκB, the inhibitor of transcription factor NF-κB, with a compound of anyone of claims 1-13.